Expression system for expressing herpesvirus glycoprotein complexes

ABSTRACT

An expression system for expressing a herpesvirus glycoprotein complex including a vector inserted with two or more nucleic acid sequences that encode two or more subunits of a herpesvirus glycoprotein complex linked by one or more linking sequences such that the subunits are co-expressed simultaneously and self-processed to assemble into a glycoprotein complex. The expression system or the vector can be included in a vaccine composition. The vaccine composition can be used for preventing or treating herpesvirus infections.

PRIORITY CLAIM

This application is a U.S. National Phase Application of InternationalApplication No. PCT/US2018/042046, filed Jul. 13, 2018, which claimspriority to U.S. Provisional Application No. 62/532,298, filed Jul. 13,2017, both of which are incorporated by reference herein in theirentirety, including drawings.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No. A103960,awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Induction of neutralizing antibodies (NAbs) that block herpesvirusglycoprotein complex-mediated entry into host cells is consideredimportant for a vaccine candidate to prevent or control herpesvirusinfection. However, using herpesvirus glycoprotein complexes as antigensis complicated by the necessity of expressing multiple subunitssimultaneously to allow efficient complex assembly and formation ofconformational neutralizing epitopes.

Although protective immune correlates of human cytomegalovirus (HCMV)are only poorly defined, induction of humoral and cellular immuneresponses targeting immunodominant antigens such as the HCMV pentamercomplex (PC), glycoprotein gB, or phosphoprotein pp65 is thought to beimportant for a vaccine candidate to prevent congenital HCMV infection.How these antigens can be assembled into a subunit vaccine toefficiently stimulate anti-HCMV immunity remains unknown.

There remains a need to inducing NAbs that effectively block herpesvirusinfections using the corresponding assembled herpesvirus glycoproteincomplexes as antigens. The technology disclosed herein satisfies thisneed.

SUMMARY

In one aspect, this disclosure relates to an expression system forexpressing a herpesvirus glycoprotein complex. The expression system mayinclude a vector inserted with two or more nucleic acid sequences thatencode two or more subunits of the herpesvirus glycoprotein complex,linked by one or more linking sequences, such that the two or moresubunits can be co-expressed simultaneously, self-cleaved andself-processed to assemble into the herpesvirus glycoprotein complex.The vector can be a plasmid vector or a viral vector. In someembodiments, the linking sequences include IRES and nucleic acidsequences encoding 2A peptides that mediate ribosomal skipping. In someembodiments, the vector is inserted with a single promoter before thetwo or more nucleic acid sequences such that the single promotercontrols the expression of the two or more nucleic acid sequences.

In another aspect, a vaccine composition for preventing herpesvirusinfection is provided. The vaccine composition may include a vectorcapable of co-expressing two or more subunits of a herpesvirusglycoprotein complex simultaneously and a pharmaceutically acceptablecarrier, adjuvant, additive or combination thereof. In some embodiments,the two or more subunits are linked by one or more linking sequences,such that the two or more subunits can be co-expressed simultaneously,self-cleaved and self-processed to assemble into the herpesvirusglycoprotein complex. In some embodiments, the linking sequences includeIRES and nucleic acid sequences encoding 2A peptides. In someembodiments, the vector is inserted with a single promoter before thetwo or more nucleic acid sequences such that the single promotercontrols the expression of the two or more nucleic acid sequences.

In another embodiment, a method of preventing herpesvirus entry into acell is provided. Such a method may include infecting the cell with aneffective amount of a viral vector, the viral vector comprising two ormore nucleic acids encoding two or more subunits of a herpesvirusglycoprotein complex, linked by one or more linking sequences.

In another embodiment, a method for preventing or treating a herpesvirusinfection in a subject is provided. Such a method may includeadministering a therapeutically effective amount of a herpesvirusvaccine to the subject, wherein the herpesvirus vaccine comprises avector capable of co-expressing two or more subunits of a herpesvirusglycoprotein simultaneously, and a pharmaceutically acceptable carrier,adjuvant, additive (e.g. CD40L) or combination thereof.

According to some of the embodiments described above, the viral vectoris a modified vaccinia Ankara (MVA) and the glycoprotein complex is HCMVpentamer complex (PC) composed of its five subunits or antigenicfragments thereof: UL128, UL130, UL131A, glycoprotein L (gL), andglycoprotein H (gH). In some embodiments, the viral vector is furtherinserted with one or more additional DNA sequences that encode one ormore additional HCMV proteins or antigenic fragments thereof. Theseadditional proteins could be either the dominant targets ofcell-mediated immunity such as pp65 and immediate early 1 and 2 proteinsor other important humoral immune targets such as glycoproteins gB, gM,gN, or gO or antigenic fragments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show construction and characterization of MVA-BAC^(TK). FIG.1A shows MVA-BAC^(TK) construction. pBeloBAC11 vector sequences (B=cat,OriS, repE, sopA/B/C, cos, loxP site) and a GFP expression cassette(GFP, Vaccinia P11 promoter) were inserted into the Thymidine kinase(TK) gene of the MVA genome utilizing ˜700 bp homologous sequences (grayfilled elements). 85 and 87=MVA ORFs 85 and (Accession Nr. U94848). FIG.1B shows BAC restriction analysis. Purified MVA-BAC^(TK) DNA wasdigested with the indicated enzymes, electrophoretically separated in a0.7% agarose gel containing ethidium bromide, and imaged by UV lightexposure. Obtained in vitro BAC restriction pattern were compared to insilico restriction pattern of MVA-BAC^(TK). FIG. 1C shows geneinsertion. An mRFP marker was inserted into MVA-BAC^(TK) into theindicated MVA deletion sites (Del2, Del3, Del6) or intergenic regionsbetween the given MVA ORFs of MVA (U94848). Panel D exemplifiesexpression of the mRFP marker after virus reconstitution. FIG. 1D showsgene expression. Viral foci of MVA-BAC^(TK)-derived virus with mRFPmarker inserted into the MVA Del2 site (panel C) were imaged byfluorescence microscopy to verify expression of the mRFP marker withinDel2 and the GFP marker of the BAC vector within the TK gene (panel A).FIG. 1E shows multi-step growth kinetics of MVA-BAC^(TK) virus. BHKcells were infected in duplicates (6 well plates) with 0.05 MOI ofMVA-BAC^(TK)-derived virus (MVA^(TK)) or wild-type MVA (MVA 1974/NIHclone 1), and virus titers (PFU/ml) were determined at the indicatedtime points.

FIGS. 2A and 2B show construction of MVA expressing P2A-linked HCMV PCsubunits. FIG. 2A shows HCMV PC subunits (gH, gL, UL128, UL130, UL131A)linked by different P2A sequences (P2A1-P2A4) were inserted either alltogether into the MVA intergenic region 69/70 (IGR69/70) of MVA-BACTK orMVA-BACDel3 to generate polycistronic vectors MVATK-PC2A1 andMVADel3-PC2A1, respectively, or separately as UL128/130/131A and gH/gLsubunit subsets into the MVA deletion 2 (Del2) site and IGR69/70 ofMVA-BACTK to generate polycistronic vector MVATK-PC2A2. B=BAC vector;mH5=Vaccinia modified H5 promoter; Del3=MVA Deletion 3 site,TK=Thymidine kinase gene. FIG. 2B shows that P2A sequences (P2A1-P2A4)with different codon usage were used to link the HCMV PC subunits withinthe MVA constructs as indicated in A. Lower 4 lines indicate DNAsequences with mutated nucleotides (marked in colors) that were used toencode for the P2A peptide between the HCMV subunits (SEQ ID NOS:2-5).Upper line shows the amino acid sequences of the P2A peptide (SEQ IDNO:1).

FIG. 3 shows expression and cleavage of P2A-linked PC subunits expressedfrom MVA vectors. BHK cells were infected with the polycistronic MVAvectors MVA^(TK)-PC2A1, MVA^(Del3)-PC2A1, and MVA^(TK)-PC2A2 (FIG. 2),control vectors MVA^(Del3)-PC or MVA^(Del3)-gB, or mock (BHK;uninfected), and HCMV PC subunits (gH, gL, UL128, UL130, UL131A) weredetected in whole cell lysates by Immunoblot using MAb and polyclonalantisera specific for the individual HCMV PC subunits. Vaccinia proteinBR5 was detected within the different samples as a loading control.kDa=kilo Dalton.

FIG. 4 shows cell surface detection of MVA expressed P2A-linked PCsubunits by NAb. BHK cells were infected with polycistronic MVA vectorMVA^(TK)-PC2A1, MVA^(Del3)-PC2A1, or MVA^(TK)-PC2A2 (FIG. 2), controlvector MVA^(Del3)-PC or MVA^(Del3)-gB, or mock (BHK; uninfected). Live,non-permeabilized cells were investigated by cell surface Flow cytometrystaining using NAb specific for quaternary conformational epitopesformed by HCMV PC subunits UL128/UL130/UL131A (1B2) or UL130/131A(54E11), or for epitopes constituted by UL128 (13B5) or gH (62-11).Following addition of primary antibodies, cells were incubated withanti-mouse Alexa Fluor 647 secondary antibody. Histogram axes representfluorescence intensity (X-axis) and cell count (Y-axis).

FIGS. 5A and 5B show NAb induction by MVA vectors expressing P2A-linkedPC subunits. FIG. 5A shows NAb titers (geometric mean titer) measuredover a 24 weeks period. Balb/c mice (N=4 to 6) were prime/boostvaccinated in three weeks interval (black rectangles) with polycistronicMVA vector MVA^(TK)-PC2A1, MVA^(Del3)-PC2A1, or MVA^(TK)-PC2A2 (FIG. 2),or control vector MVA^(Del3)-PC. At week 0 (pre-immune serum), 3, 7, 13,and 24, HCMV specific NAb titer (log 10 NT50) in mouse sera weremeasured against HCMV strain TB40/E on ARPE-19 EC (continuous lines) oron MRC-5 FB (dashed lines). The dotted line indicates the limit of NAbdetection. Bars represent 95% confidence intervals. FIG. 5B showsstatistical analysis of NAb titers. Wilcoxon matched-pairs test was usedto investigate differences of ARPE-19 EC and MRC-5 FB specific NAbtiters at week 7 and week 24 in vaccine groups immunized with theindicated MVA vectors. P values less than 0.05 were indicated with *.

DETAILED DESCRIPTION

Expression systems, vectors, vaccines for use in preventing or treatinghuman herpesvirus infections are provided herein. The expressionsystems, vectors and vaccines, which are described in detail below,generate neutralizing antibodies (NAb) against human herpesvirusantigenic proteins or fragments to block entry of the human herpesvirusglycoprotein complex-mediated entry into host cells, thereby preventinghorizontal and vertical virus transmission. This disclosure relates tothe simultaneous expression of two or more herpesvirus glycoproteincomplex subunits utilizing a linking sequence between the subunits toco-express self-processing polyproteins that efficiently assemble intoprotein complexes. The expressed subunits can encode for glycoproteinsof any of the known human herpesvirus, including cytomegalovirus (CMV),varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Kaposi'sSarcoma-associated herpesvirus (KSHV), herpes simplex virus type 1(HSV-1), herpes simplex virus type 2 (HSV-2), human herpesvirus 6(HHV-6), human herpesvirus 7 (HHV-7), or any other herpesvirus thatinfects vertebrates or invertebrates.

In some embodiments disclosed herein, the glycoprotein complex subunitscan be either expressed by plasmid vectors such as pcDNA, pTT5, pCAGGSor related vectors, or viral vectors such as CMV, Vaccinia, ModifiedVaccinia Ankara (MVA), Adenovirus, Sindbis virus, or related RNA orDNA-based viral vectors. The individual subunits can be linked bycleavage sequences such that the co-expressed subunits can beself-cleaved and self-assembled into glycoprotein complexes.

In some embodiments, the expression systems, vectors, vaccines describedherein include one or more expression cassettes, each of which includesa single promoter and a sequence that encodes two or more herpesvirusglycoprotein complex subunits. As a result, the two or more herpesvirusglycoprotein complex subunits are co-expressed simultaneously, i.e.,under control of a single promoter, obviating the need for multiplepromoters or vectors. In certain embodiments, each expression cassetteincludes two, three, four, five, or even higher numbers of herpesvirusglycoprotein complex subunits, the expression of which are under controlof a single promoter. In other embodiments, each expression cassetteincludes more than ten herpesvirus glycoprotein complex subunits. Insome embodiments, a vector may include more than one such expressioncassette.

In some embodiments, internal ribosome entry sites (IRES) can beintroduced in between nucleic acid sequences encoding two or moreherpesvirus glycoprotein complex subunits that are co-expressed,flanking the sequences encoding the two or more subunits. Although IREScan be used to link the expression of multiple genes under a singlepromoter, the use of multiple IRES sequences might be limited by sizeconstraints or instability due to its relatively larger size comparingto 2A signal sequences. In some embodiments, 2A signal sequences thatencode for the 2A peptide of food-and-mouth disease virus (F2A), equinerhinitis A virus (E2A), porcine teschovirus-1 (P2A), Thoseaasigna virus(T2A), cytoplasmic polyhedrosis virus (BmCPV 2A), or flacherie virus(BmlFV 2A) can be used to link multiple genes under a single promoter.2A signal sequences have been found in picomaviruses, insect viruses andtype C rotaviruses. Various suitable eukaryotic cell promoters can beused, including but not limited to, immediate-early I promoter of humanCMV or the chicken beta actin promoter, promoters of vaccinia virus(mH5, pSyn, P11, p7.5), etc.

Additionally, a furin cleavage site preceding the 2A signal sequencescan be incorporated to remove the 2A peptides following self-processingof the 2A-linked polyproteins. Furin is an enzyme that occurs in theGolgi apparatus and cleaves at very short signal peptides such as KKKRor RKKR motif. Furin cleavage contributes to protein processing andmaturation. These short signal peptides can be added to the N-terminusof the 18-22 amino acid long 2A skipping signals so that they areremoved following 2A-mediated processing of the herpesvirusglycoproteins, except for one or two remaining amino acids. Theresultant product can be even more “native.” Although it is preferredthat the 2A-linked subunits are expressed all from one vector throughthe use of one or more expression cassettes, it is also possible toexpress the 2A-linked subunits from two or more separate vectors. As anexample for this system, the P2A skipping system and a novel bacterialartificial chromosome (BAC) clone of the clinically deployable MVAvector were used to induce CMV NAb by self-processing subunits of theCMV pentamer complex. In some embodiments, using markerless BACmanipulation, codon-optimized and P2A-linked PC subunits can be insertedinto MVA either into one insertion site to generate MVA-PC2A1, or asUL128/130/131A and gH/gL subunit subsets into two separate insertionsites to generate MVA-PC2A2. As detailed in the working examples, whileall PC subunits were expressed in significantly higher levels fromMVA-PC2A2 compared to MVA-PC2A1, the PC subunits of both polycistronicvectors were efficiently cleaved and transported to the cell surface asprotein complexes forming conformational and linear neutralizingepitopes. In addition, vaccination of mice with two doses of either ofthe vaccine vectors resulted in potent and comparable HCMV specific NAbresponses that remained stable for at least six months.

Despite the current Zika virus (ZIKV) outbreak in the Americas and itsassociation with a surge of microcephaly, human cytomegalovirus (HCMV)remains the most common infectious cause of permanent birth defectsworldwide (5, 31). While there are currently no vaccines that couldprotect pregnant women and their developing fetuses from either HCMV orZIKV infection, vaccine development for HCMV unlike that for ZIKV spansalmost a period of more than five decades (31, 36, 38). Many HCMVvaccine candidates have been preclinically and clinically evaluated,though encouraging findings for feasibility of a congenital HCMV vaccinehave been obtained only with an approach based on envelope glycoproteingB, a central mediator in HCMV host cell entry and important humoralimmune target (30). In phase II clinical trials, gB adjuvanted with MF59has been shown to afford 43 to 50% efficacy to prevent primary infectionof HCMV seronegative (HCMV⁻) young women or adolescent girls (3, 37).While these efficacy rates are considered insufficient for vaccinelicensure, these findings hold promise that a vaccine candidate withimproved immunogenicity to elicit humoral and cellular immune responsescould provide levels of protection that significantly alter the outcomeof congenital HCMV infection.

Although it remains unclear why previous vaccine candidates have failedto effectively prevent HCMV infection, one explanation for their failurecould be their inability to elicit certain types of neutralizingantibodies (NAb) (11). Over the last years it has been recognized thatHCMV host cell infection occurs by two virus entry routes that areblocked by NAb of varying specificity and potency (29, 40). While HCMVinfection of all susceptible cell types appears to depend on envelopeglycoproteins gB and gH/gL/gO, the pentamer complex (PC) composed of gH,gL, UL128, UL130, and UL131A is additionally required for virus entryinto many biologically relevant host cells including epithelial cells(EC), but dispensable for virus entry into other important cells such asfibroblasts (FB) (1, 20, 22, 50, 51, 55, 59, 64). Important for vaccinedesign, NAb predominantly recognizing quaternary conformational epitopesof the PC are substantially more potent than NAb interfering with gB andgH/gL/gO entry function (7, 17, 29). Since HCMV NAb were almostexclusively correlated with measurements based on FB before theidentification of anti-PC antibodies, previous vaccine strategies suchas those employing only gB were not designed to account for the potentNAb responses blocking EC entry (11, 18). Overall these findings suggestthat inclusion of the PC into a vaccine candidate could eliminate acaveat of previous HCMV vaccines in eliciting NAb and, hence, lead toprotection efficacy higher than that achievable with gB alone.

While there are only a few findings that support an important protectiverole of NAb targeting the PC (26, 27), many preclinical vaccine conceptsbased on the PC have been developed to stimulate HCMV NAb responses (16,23, 58, 60, 62). These vaccine approaches consistently demonstrate thatthe PC is superior to gB and gH/gL in inducing NAb that prevent ECinfection. A recently developed PC-specific vaccine concept was based onthe well-characterized and clinically well-tolerated Modified VacciniaAnkara (MVA) vector (7, 60, 62). In addition to its excellent safetyrecord (19, 52), MVA provides many advantages over other vector systemsincluding a large capacity for heterologous antigens, ability to elicitrobust antigen specific immunity, and a versatile cytoplasmic expressionsystem that allows efficient antigen delivery without the risk for viralDNA integration into host chromosomes (6, 9, 15). By utilizing MVAbacterial artificial chromosome (BAC) technology, multiple MVA insertionsites can be used to generate a single MVA vector co-expressing all fiveHCMV PC subunits (60). For example, the heterologous sequences can beinserted into TK site, one of the six major deletion sites (Del1-6), orMVA intergenic regions (there are over 180 MVA genes), or non-essentialgenes to drive gene expression by intrinsic promoter elements. Thisvector, termed MVA-PC, stimulated potent and durable HCMV specific NAbresponses in mice and rhesus macaques (RM). However, while effective ineliciting NAb, MVA-PC appeared limited in accommodating additional HCMVantigens to further enhance its ability to induce anti-HCMV immuneresponses due to the complexity of the vector design to co-express allfive HCMV PC subunits (60).

By exploiting the ribosomal skipping mechanism conferred by 2A peptides(14, 39), an approach of expressing the five HCMV PC subunits from MVAas only one or two self-processing polyproteins is disclosed herein. The2A ribosomal skipping system is widely-used to express multi-proteincomplexes due to the relative small sizes of 2A peptides (18-22 aminoacids) and because it allows stoichiometric expression of the individual2A-linked subunits (12, 24, 45). As demonstrated in the workingexamples, by utilizing a novel BAC of MVA, codon-optimized andP2A-linked DNA sequences of the five PC subunits were inserted into MVAeither all together into only one insertion site or as UL128/130/131Aand gH/gL subunit subsets into two separate insertion sites, resultingin MVA-PC2A1 and MVA-PC2A2, respectively. Whereas expression levels ofall five PC subunits were significantly higher with MVA-PC2A2 than withMVA-PC2A1, the PC subunits expressed from both vaccines were efficientlycleaved and transported to the cell surface as five member proteincomplexes that formed conformational neutralizing epitopes. In addition,vaccination of mice with two doses of either MVA-PC2A1 or MVA-PC2A2resulted in induction of potent and durable HCMV NAb responses. Thisapproach of eliciting NAb by self-processing PC polyproteinssignificantly reduces the complexity of simultaneously co-expressing allfive PC subunits, which could be useful for many other vector systems toefficiently express the PC and stimulate HCMV specific NAb and may serveas a template to induce NAb by multi-protein glycoprotein complexes ofother viruses.

Since the discovery of the HCMV PC and its recognition as a target ofpotent NAb responses that prevent in vitro HCMV infection of manybiologically-relevant host cells (7, 29, 55), the HCMV PC has become amajor focus in HCMV vaccine development (16, 23, 58, 60, 62). As most ofthese potent NAb recognize quaternary conformational epitopes formed bymore than one subunit of the PC (7, 29), vaccine-mediated NAb inductionbased on the PC relies on simultaneous co-expression of all five PCsubunits to enable efficient subunits assembly and formation ofconformational neutralizing epitopes. While the large insertion capacityand versatile expression system of MVA are advantageous (60, 62), othershave mastered it by employing replication defective HCMV (16) or viralvector or plasmid expression constructs with multiple promoter elementsor internal ribosomal entry sites (IRES) (21, 28, 58). In addition,Kabanova and colleagues developed a vaccine approach based on purifiedPC protein that was generated via 2A-linked expression constructs;however details of the vector construction to produce the purified PCprotein remained unclear (23).

Disclosed herein is an expression system based on ribosomal skippingmechanism, i.e., by P2A peptides of porcine teschovirus-1 to induce HCMVNAb by MVA vectors expressing self-processing PC subunits inserted intoonly one or two MVA insertion sites (FIG. 2). Because of the relativelysmall sequence of the P2A peptides and the use of identical 2A peptidesfor polyprotein cleavage (FIG. 2) (24, 45), this system can be used forother vector systems to efficiently express the PC and to stimulate HCMVNAb and may form a template for stimulating NAb by multi-subunitglycoprotein complexes of other viruses as well. Although PC andinducing HCMV NAb are used as examples in this disclosure, expressionand assembly of other herpesvirus glycoprotein complexes can be achievedbased on this disclosure.

Besides introducing an approach for stimulating potent NAb byself-cleavable PC subunits, also disclosed herein is a novel BAC cloneof MVA that can serve as a powerful tool to develop clinicallydeployable vaccine vectors for infectious diseases and cancer (FIG. 1).While the conventional way of producing recombinant MVA by homologousrecombination in eukaryotic cells may be as efficient as the BACtechnology to generate antigenically less complex MVA vectors, the BACtechnology appears to be in particular useful to generate MVA withmultiple antigen insertions due to the possibility of repeatedly andseamlessly manipulate BAC DNA by highly-efficient and versatilemutagenesis techniques (8, 10, 60). In contrast to two previouslygenerated MVA-BAC—including original MVA-BAC^(Del3)—(8, 35), theconstruction of MVA-BAC^(TK) was based on the insertion of BAC vectorsequences into the TK gene locus. As demonstrated in the workingexamples, the BAC restriction and sequencing analysis as well as growthkinetics of BAC-derived virus suggest that MVA-BAC^(TK) comprises anintact, full-length genomic clone of MVA. In addition, the comparableand potent HCMV specific NAb responses induced by the polycistronic MVAvectors derived from MVA-BAC^(TK) and the immunologicallywell-characterized original MVA-BAC^(Del3) indicate that MVA-BAC^(TK)has excellent immunogenicity properties to stimulate antigen specificimmunity. Accordingly, the MVA-BAC^(TK) disclosed herein can be used fordeveloping antigenically complex recombinant MVA vaccine vectors.

The MVA-BAC^(TK)-derived polycistronic MVA vectors expressingself-processing HCMV PC subunits disclosed herein can be used fordeveloping a multi-component MVA vaccine candidate to prevent congenitalHCMV infection. By linking the PC subunits together via P2A peptidesallowing expression of all five PC subunits using only one or two MVAinsertion sites, the complexity of the MVA vector construction isreduced significantly compared to MVA^(Del3)-PC to simulate potent HCMVNAb responses (60). As the developed MVA vectors expressing P2A-linkedPC subunits are as potent as MVA^(Del3)-PC to stimulate HCMV NAb inmice, it can be anticipated that the polycistronic MVA vectors will alsoelicit NAb in non-human primates considering our previous findings withMVA^(Del3)-PC (60). As a consequence of reducing the insertion of the PCsubunits to only one or two MVA insertion sites, other commonly used MVAinsertion sites remain available for introducing additional HCMVantigens such as gB and pp65 to further enhance the ability of thedeveloped polycistronic MVA vectors to stimulate anti-HCMV humoral andcellular immune responses. In addition, non-commonly used MVA insertionsites can be used for additional antigen insertion based on thedemonstration of inserting mRFP into 12 different insertion sitesdistributed over the cloned MVA genome of MVA-BAC^(TK) (FIG. 1).

Despite expressing the PC subunits of the different MVA vectors by thesame vaccinia promoter (mH5), MVA^(TK)-PC2A2 with PC subunit subsetsinserted into two separate insertion sites expressed significantly loweramounts of the HCMV proteins compared to MVA^(Del3)-PC, and lowest PCsubunit expression was observed with the pentacistronic vectorsMVA^(TK)-PC2A1 and MVA^(Del3)-PC2A1 (FIG. 3). While it appeared that thepentacistronic vectors did not express detectable amounts of UL131A byImmunoblot, the cell surface Flow Cytometry staining analysis usingantibodies to conformational epitopes constituted by UL128/130/131A andUL130/131A provide evidence that these vectors expressed also UL131A inaddition to all other four PC subunits (FIG. 4). While the reason forthe variable PC subunit expression levels between the MVA vectors remainunknown, they appear to be associated with the length of the insertedsequences, which may lead to different transcription or translationefficacy or differences in post transcriptional and translationprocessing of the PC subunits. Equally as likely, the co-translationalP2A skipping mechanism may impair processing of the nascentself-processing PC polyproteins, and the higher the number of cleavagesignals within the expression construct the less efficient theexpression. However, the differences in PC subunit expression levelsbetween the three polycistronic vectors and MVA^(Del3)-PC may also be aresult of different codon-usage, since the self-processing PC subunitsof the polycistronic MVA vectors were encoded by codon-optimized DNAsequences while the PC subunits of MVA^(Del3)-PC were encoded by DNAsequences identical to those present in HCMV TB40/E.

Considering the marked differences in PC subunit expression of thepolycistronic MVA vectors and MVA^(Del3)PC, it was unexpected that thesevectors stimulated comparable HCMV specific NAb responses (FIGS. 3 and5). This discrepancy in PC subunit expression and PC-specific NAbinduction could be explained by the observations made for cell surfacedetection of the PC subunits. While cellular PC subunit expressionlevels of the different vectors were significantly different byImmunoblot (FIG. 3), comparable cell surface Flow cytometry stainingintensity of the PC subunits using different NAb was observed for allMVA vectors (FIG. 4), suggesting that the PC subunits expressed from thepolycistronic MVA vectors and MVA^(Del3)PC were transported to the cellsurface with similar efficacy. This could explain why the differentvectors had similar ability to stimulate HCMV specific NAb, despiteexpressing different amounts of HCMV subunits. Consequently, only verylow expression levels of the PC subunits appear to be required forstimulating anti-HCMV NAb responses, at least when considering NAbinduction in mice. MVA^(TK)-PC2A2 and MVA^(Del3)-PC may thereforeoverexpress a proportion of the PC subunits, potentially resulting ininefficient processing or assembly of the PC subunits. In this context,comparing NAb induction by the different MVA vectors in mice by dosede-escalation may help to distinguish whether the MVA vectors havedifferent ability to stimulate HCMV NAb. As all three polycistronic MVAvectors stimulated potent NAb responses in mice, they all represent abasis to develop a multi-component MVA vaccine vector for preventingcongenital HCMV infection.

While there are many innovative vaccine candidates to mitigatecongenital HCMV infection, of which one has recently entered phase Iclinical testing (54), the approach for developing a congenital HCMVvaccine candidate disclosed herein is attractive for the followingreasons. First, it is based on the highly-attenuated MVA vaccine vectorthat has been tested safely on over 120000 people in Europe (19, 52),indicating that the vaccine approach will be clinically deployable.Second, MVA is well-known to elicit robust antigen specific humoral andcellular immune responses (9, 15), suggesting that the approach wouldallow to effectively induce HCMV immune responses by multiple antigens.This is supported by the recently published phase I clinical results foran MVA vector expressing immunodominant T cell targets pp65 and IE1/2that induced potent and durable antigen specific cellular immuneresponses in healthy adults (25). Third, it is based on vaccineconstruction using BAC technology that will allow to effectively exploitthe large insertion capacity and versatile expression system of MVA toassemble multiple HCMV antigens including the PC, gB, and pp65 into asingle MVA vector (8, 60). Fourth, it is based on the expression of amembrane tethered PC that allows induction of potent HCMV specific NAbresponses in mice and RM by only two immunizations (60). For thesereasons, the vaccine approach disclosed herein may represent a uniquestrategy to develop a multi-component vaccine to mitigate congenial HCMVdisease.

Based on the studies described above, expression systems, viral vectorsand vaccines that may be used in methods for inhibiting of herpesvirusentry into host cells have been developed and described herein.

Expression Systems, Vectors and Vaccines

According to the embodiments described herein, a herpesvirus antigenicprotein expression system (or “antigen expression system”) is providedherein. In one embodiment, the antigen expression system may include acloning vector to clone an expression vector that is able to express oneor more herpesvirus antigenic proteins or antigenic fragments thereof.In some embodiments, the antigenic proteins or antigenic fragmentsthereof are herpesvirus glycoprotein complex, subunits thereof, orantigenic fragments of one or more subunits. The herpesvirusglycoprotein complex subunits or antigenic fragments thereof are derivedfrom cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barrvirus (EBV), Kaposi's Sarcoma-associated herpesvirus (KSHV), herpessimplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), humanherpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), or any otherherpesvirus that infects vertebrates or invertebrates.

In one embodiment, the cloning vector is a BAC, which is a DNA constructthat may be used to clone one or more target herpesvirus genes bytransformation in bacteria (e.g., E. coli). The use of BAC as a cloningvector allows for stable cloning of very large DNA sequences, and can beeasily manipulated using genetic techniques established for E. coli. Insome embodiments, the BAC cloning vector is used to clone an expressionvector. The expression vector may be a plasmid, a BAC, a viral vector(e.g., adenoviral vectors, adeno-associated viral vectors, RNA viralvectors, lentiviral vectors or retroviral vectors), a viral vectorconstructed as a BAC, or any other suitable vector that is able toexpress a recombinant protein, a viral vector or both.

In some embodiments, the expression vector (e.g., the viral vector) iscapable of expressing one or more immunogenic or antigenic herpesvirusproteins or functional fragments thereof. An immunogenic protein is aprotein that, when introduced to a subject, is recognized by thesubject's immune cells, thereby stimulating an immune reaction. Theimmune reaction may result in antibody production (e.g., neutralizingantibody production) against that protein. A functional or antigenicfragment of an immunogenic protein is any portion of the protein thatcontains an antigenic portion of the protein or is an antigenic portionof the protein which may contain at least one epitope. In someembodiments, the one or more immunogenic proteins or functionalfragments thereof may be an immunogenic protein complex, which includesa set of immunogenic protein subunits or functional fragments thereof.

In one embodiment, the BAC cloning vector is used to clone a viralexpression vector. In such embodiments, the BAC vector is inserted intothe genome of the viral expression vector to generate a virus-BACconstruct or plasmid. A bacterial host (e.g., E. coli) is thentransfected with the virus-BAC plasmid to clone the viral vector.Transfection of the virus-BAC clones into eukaryotic cells susceptibleto infection by the viral vector results in reconstitution of therecombinant virus. The resulting reconstituted viral vectors may then beused to infect target tissues or cells in a host.

In some embodiments, the glycoprotein complex subunits can be expressedby plasmid vectors such as pcDNA, pTT5, pCAGGS or related vectors. Insome embodiments, the expression vector can be a viral vector derivedfrom any suitable adenovirus, sindbis virus, CMV, or poxvirus including,but not limited to, Avipoxvirus (e.g., canarypox virus and relatedstrains such as ALVAC; fowipox virus), Orthopoxvirus (e.g., vacciniavirus strains such as the Western Reserve or Lister strain, Copenhagenstrain (NYVAC), Dryvax strain, modified vaccinia Ankara (MVA) strain,ACAM1000, and ACAM2000 strain), Parapoxvirus (e.g., Orf virus). In oneembodiment, the viral vector is a modified vaccinia Ankara (MVA), whichis cloned into the BAC cloning vector (“MVA-BAC”) and is able to expressone or more immunogenic herpesvirus proteins or antigenic fragmentsthereof. Any suitable MVA strain may be cloned by a BAC in accordancewith the embodiments described herein, including, but not limited to the1974-MVA strain, VR strain or ACAM 3000 strain.

In one embodiment, one or more immunogenic herpesvirus proteins orantigenic fragments thereof are HCMV glycoproteins including a set ofimmunogenic protein subunits or functional fragments thereof that arepart of a CMV pentamer complex (PC). The CMV pentamer complex is a HCMVprotein complex that includes the following five immunogenic proteinsubunits or functional fragments thereof: UL128, UL130, UL131A, gL, andgH. Co-expression of all five of the PC subunits is required in singlecells to obtain functional expression. Therefore, a single deliveryvector is needed, since there is no current generally acceptableapproach to guide >1 individual DNA or viral vectors to assemble aprotein complex in vivo by co-expression of all 5 PC components.

Simultaneous co-expression of the PC complex that includes the UL128,UL130, UL131A, gL, and gH proteins or antigenic fragments thereof by theexpression systems and viral vectors described herein results instimulation of neutralizing antibodies (NAb) by a hosts immune systemthat block HCMV infection in susceptible cells such as epithelial andendothelial cells.

In other embodiments, the expression vector may include additional HCMVproteins including, but not limited to, pp65, gB, IE1 gM, gN, gO, andother suitable antigenic HCMV proteins known in the art. Theseadditional genes may be inserted into a first expression vector with thePC subunits, or alternatively, may be inserted into a second expressionvector to be administered in combination with the first expressionvector. In some embodiments, all subunits are inserted into the sameinsertion site of an MVA-BAC vector, such as the TK insertion site. Inother embodiments, one or more subunits are inserted into two or moredifferent insertion sites of an MVA-BAC vector.

According to the embodiments described herein, an immunization regimenis provided. The immunization regimen may include plasmids, viralvectors, live-attenuated viruses, purified protein, or virus-likeparticles that express or comprise the herpesvirus glycoprotein complexsubunits. The immunization regimen may be administered via prime/boosthomologous (e.g. using only the same vaccine type) or heterologous (e.gusing different vaccine types) vaccination. The immunization regimen maybe administered in a dose vaccination schedule involving two or moreimmunizations, which may be administered 2 weeks to 6 month apart.

In other embodiments, the MVA vector described above may be a primingimmunization. In such a case, the aforementioned primes can also be usedas booster vectors after one or more (e.g., one, two, three, four, ormore) consecutive MVA immunizations. Alternatively, priming and boostingvectors can alternate such that the heterologous immunization willinclude an MVA or alternate vector as a prime followed by MVA or analternate vector as a boost from 1 to 4 times as an example. Othersuitable immunization schedules or regimens that are known in the artmay be used according to the embodiments described herein by thoseskilled in the art.

According to some embodiments, the nucleic acid sequences encoding twoor more subunits of a herpesvirus glycoprotein complex are assembledinto a single vector, with a linking sequence inserted between thenucleic acid sequences encoding two or more subunits. For example, CMVpentamer complex subunits may be linked through linking sequences suchas internal ribosome entry sites (IRES), derived from a number ofdifferent RNA viruses that are well known in the art and sequencesencoding 2A peptides, to link all or a portion of the subunits in oneinsertion site or multiple insertion sites. The 2A signal sequenceencoding a 2A peptide of foot-and-mouth disease virus (F2A), a 2Apeptide of equine rhinitis A virus (E2A), a 2A peptide of porcineteschovirus-1 (P2A), a 2A peptide of cytoplasmic polyhedrosis virus(BmCPV 2A), a 2A peptide of flacherie virus (BmlFV 2A), or a 2A peptideof Thosea asigna virus (T2A), can be used.

A recombinant vector, such as the MVA viral vector described above; orany other suitable alternative vector including suitable primer orbooster vectors described above, may be part of a herpesvirus vaccinecomposition that may be used in methods to treat or prevent herpesvirusinfection. The vaccine composition as described herein may comprise atherapeutically effective amount of a recombinant viral vector asdescribed herein, and further comprising a pharmaceutically acceptablecarrier according to a standard method. Examples of acceptable carriersinclude physiologically acceptable solutions, such as sterile saline andsterile buffered saline.

In some embodiments, the vaccine or pharmaceutical composition may beused in combination with a pharmaceutically effective amount of anadjuvant to enhance the anti-CMV effects. Any immunologic adjuvant thatmay stimulate the immune system and increase the response to a vaccine,without having any specific antigenic effect itself may be used as theadjuvant. Many immunologic adjuvants mimic evolutionarily conservedmolecules known as pathogen-associated molecular patterns (PAMPs) andare recognized by a set of immune receptors known as Toll-like Receptors(TLRs). Examples of adjuvants that may be used in accordance with theembodiments described herein include Freund's complete adjuvant,Freund's incomplete adjuvant, double stranded RNA (a TLR3 ligand), LPS,LPS analogs such as monophosphoryl lipid A (MPL) (a TLR4 ligand),flagellin (a TLR5 ligand), lipoproteins, lipopeptides, single strandedRNA, single stranded DNA, imidazoquinolin analogs (TLR7 and TLR8ligands), CpG DNA (a TLR9 ligand), Ribi's adjuvant (monophosphoryl-lipidA/trehalose dicorynoycolate), glycolipids (α-GalCer analogs),unmethylated CpG islands, oil emulsion, liposomes, virosomes, saponins(active fractions of saponin such as QS21), muramyl dipeptide, alum,aluminum hydroxide, squalene, BCG, cytokines such as GM-CSF and IL-12,chemokines such as MIP 1-α and RANTES, activating cell surface ligandssuch as CD40L, N-acetylmuramine-L-alanyl-D-isoglutamine (MDP), andthymosin α1. The amount of adjuvant used can be suitably selectedaccording to the degree of symptoms, such as softening of the skin,pain, erythema, fever, headache, and muscular pain, which might beexpressed as part of the immune response in humans or animals after theadministration of this type of vaccine.

In further embodiments, use of various other adjuvants, drugs oradditives with the vaccine of the invention, as discussed above, mayenhance the therapeutic effect achieved by the administration of thevaccine or pharmaceutical composition. The pharmaceutically acceptablecarrier may contain a trace amount of additives, such as substances thatenhance the isotonicity and chemical stability. Such additives should benon-toxic to a human or other mammalian subject in the dosage andconcentration used, and examples thereof include buffers such asphosphoric acid, citric acid, succinic acid, acetic acid, and otherorganic acids, and salts thereof; antioxidants such as ascorbic acid;low molecular weight (e.g., less than about 10 residues) polypeptides(e.g., polyarginine and tripeptide) proteins (e.g., serum albumin,gelatin, and immunoglobulin); amino acids (e.g., glycine, glutamic acid,aspartic acid, and arginine); monosaccharides, disaccharides, and othercarbohydrates (e.g., cellulose and derivatives thereof, glucose,mannose, and dextrin), chelating agents (e.g., EDTA); sugar alcohols(e.g., mannitol and sorbitol); counterions (e.g., sodium); nonionicsurfactants (e.g., polysorbate and poloxamer); antibiotics; and PEG.

The vaccine or pharmaceutical composition containing a recombinant viralvector described herein may be stored as an aqueous solution or alyophilized product in a unit or multiple dose container such as asealed ampoule or a vial.

Preventing Herpesvirus Entry into a Cell, Treating and PreventingHerpesvirus Infection

The antigen expression system described above may be used in in vitro,in vivo or ex vivo methods of preventing herpes virus entry into a cellor a population of cells. In some embodiments, methods for preventingherpesvirus entry into a cell or a population of cells include steps ofinfecting the cell or population of cells with an effective amount of aviral vector capable of expressing a herpesvirus glycoprotein complex orantigenic fragments thereof.

In other embodiments, methods for treating or preventing a herpesvirusinfection in a subject are provided. Such methods may includeadministering a therapeutically effective amount of a herpesvirusvaccine to the subject. The herpesvirus vaccine may include at least oneactive ingredient, wherein the at least one active ingredient includes aviral vector that is capable of expressing a herpesvirus glycoproteincomplex or antigenic fragments thereof, such as those described herein.

The expression systems, vectors and vaccines described herein may beused to treat or prevent any herpesvirus infection. For example, HCMVinfection that infects epithelial cells, endothelial cells, fibroblastsor a combination thereof can be treated or prevented. Examples of HCMVinfections that may be treated or prevented using the methods describedherein may include, but is not limited to, congenital HCMV infection,opportunistic HCMV infections in subjects with compromised immune system(e.g., organ and bone marrow transplant recipients, cancer patients andchemotherapy recipients, patients receiving immunosuppressive drugs andHIV-infected patients) and silent HCMV infections in otherwise healthysubjects.

The term “effective amount” as used herein refers to an amount of acomposition that produces a desired effect. For example, a population ofcells may be infected with an effective amount of a viral vector tostudy its effect in vitro (e.g., cell culture) or to produce a desiredtherapeutic effect ex vivo or in vitro. An effective amount of acomposition may be used to produce a therapeutic effect in a subject,such as preventing or treating a target condition, alleviating symptomsassociated with the condition, or producing a desired physiologicaleffect. In such a case, the effective amount of a composition is a“therapeutically effective amount,” “therapeutically effectiveconcentration” or “therapeutically effective dose.” The preciseeffective amount or therapeutically effective amount is an amount of thecomposition that will yield the most effective results in terms ofefficacy of treatment in a given subject or population of cells. Thisamount will vary depending upon a variety of factors, including but notlimited to the characteristics of the composition (including activity,pharmacokinetics, pharmacodynamics, and bioavailability), thephysiological condition of the subject (including age, sex, disease typeand stage, general physical condition, responsiveness to a given dosage,and type of medication) or cells, the nature of the pharmaceuticallyacceptable carrier or carriers in the formulation, and the route ofadministration. Further an effective or therapeutically effective amountmay vary depending on whether the composition is administered alone orin combination with another composition, drug, therapy or othertherapeutic method or modality. One skilled in the clinical andpharmacological arts will be able to determine an effective amount ortherapeutically effective amount through routine experimentation, namelyby monitoring a cell's or subject's response to administration of acomposition and adjusting the dosage accordingly. For additionalguidance, see Remington: The Science and Practice of Pharmacy, 21^(st)Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams &Wilkins, Philadelphia, Pa., 2005, which is hereby incorporated byreference as if fully set forth herein.

“Treating” or “treatment” of a condition may refer to preventing thecondition, slowing the onset or rate of development of the condition,reducing the risk of developing the condition, preventing or delayingthe development of symptoms associated with the condition, reducing orending symptoms associated with the condition, generating a complete orpartial regression of the condition, or some combination thereof.Treatment may also mean a prophylactic or preventative treatment of acondition.

In some embodiments, the vaccine or pharmaceutical composition describedherein may be used in combination with other known pharmaceuticalproducts, such as immune response-promoting peptides and antibacterialagents (synthetic antibacterial agents). The vaccine or pharmaceuticalcomposition may further comprise other drugs and additives. Examples ofdrugs or additives that may be used in conjunction with a vaccine orpharmaceutical composition described herein include drugs that aidintracellular uptake of the recombinant virus or MVA or recombinanttransgenic protein of the present invention, liposome and other drugsand/or additives that facilitate transfection, (e.g., fluorocarbonemulsifiers, cochleates, tubules, golden particles, biodegradablemicrospheres, and cationic polymers).

In some embodiments, the amount of the active ingredient contained inthe vaccine or pharmaceutical composition described herein may beselected from a wide range of concentrations, Virus Particle Unit (VPU),Plaque Forming Unit (PFU), weight to volume percent (w/v %) or otherquantitative measure of active ingredient amount, as long as it is atherapeutically or pharmaceutically effective amount. The dosage of thevaccine or pharmaceutical composition may be appropriately selected froma wide range according to the desired therapeutic effect, theadministration method (administration route), the therapeutic period,the patient's age, gender, and other conditions, etc.

In some aspects, when a recombinant viral vector is administered to ahuman subject as an active ingredient of the vaccine or pharmaceuticalcomposition, the dosage of the recombinant virus or MVA may beadministered in an amount approximately corresponding to 10² to 10¹⁴PFU, preferably 10⁵ to 10¹² PFU, and more preferably 10⁵ to 10¹⁰ PFU perpatient, calculated as the PFU of the recombinant virus.

In further aspects, when a recombinant viral vector is administered to asubject as an active ingredient of the vaccine or pharmaceuticalcomposition, the dosage may be selected from a wide range in terms ofthe amount of expressible DNA introduced into the vaccine host or theamount of transcribed RNA. The dosage also depends on the strength ofthe transcription and translation promoters used in any transfer vectorsused.

In some embodiments, the vaccine composition or pharmaceuticalcomposition described herein may be administered by directly injecting arecombinant viral vector suspension prepared by suspending therecombinant virus or MVA in PBS (phosphate buffered saline) or salineinto a local site (e.g., into the lung tissue, liver, muscle or brain),by nasal or respiratory inhalation, or by intravascular (i.v.) (e.g.,intra-arterial, intravenous, and portal venous), subcutaneous (s.c.),intracutaneous (i.c.), intradermal (i.d.), or intraperitoneal (i.p.)administration. The vaccine or pharmaceutical composition of the presentinvention may be administered more than once. More specifically, afterthe initial administration, one or more additional vaccinations may begiven as a booster. One or more booster administrations can enhance thedesired effect. After the administration of the vaccine orpharmaceutical composition, booster immunization with a pharmaceuticalcomposition containing the recombinant virus or MVA as described hereinmay be performed.

The following examples are intended to illustrate various embodiments ofthe invention. As such, the specific embodiments discussed are not to beconstructed as limitations on the scope of the invention. It will beapparent to one skilled in the art that various equivalents, changes,and modifications may be made without departing from the scope ofinvention, and it is understood that such equivalent embodiments are tobe included herein. Further, all references cited in the disclosure arehereby incorporated by reference in their entirety, as if fully setforth herein.

EXAMPLES Example 1: Materials and Methods

Viruses and Cells:

Baby hamster kidney (BHK-21) cells, chicken embryo fibroblasts (CEF),ARPE-19 EC, and MRC-5 FB were maintained and propagated as describedpreviously (56, 57, 60). BHK-21, ARPE-19, and MRC-5 cells were obtainedfrom the American Type Culture Collection (ATCC CCL-10 and ATCCCRL-2302). Chicken anemia virus-negative CEF cells were purchased fromCharles River (Order Nr. 1010087). MVA 1974/NIH clone 1 and Fowl poxvirus HP1.441 were kindly provided by Bernard Moss (NIAID) (33). HCMVTB40/E expressing green fluorescent protein (GFP) was reconstituted fromTB40/E BAC, a kind gift from Christian Sinzger (Ulm University, Germany)(41, 44). For generating MVA stocks, MVA was propagated in BHK cells andpurified through 36% sucrose density ultracentrifugation, thenre-suspended in 1 mM Tris-buffered saline and stored at −80° C. (56,57). Titer of MVA stocks were determined on BHK cells by immunostainingof viral foci 18-24 after infection using rabbit polyclonal antibodiesto vaccinia virus. TB40/E stocks were produced following viruspropagation in ARPE-19 cells as described (60). Briefly, 70-90%confluent ARPE-19 cells were infected with TB40/E at 0.1 multiplicity ofinfection (MOI) and re-seeded weekly until 90-100% of cells wereinfected as monitored by GFP expression. Virus particles wereconcentrated from clarified medium by ultracentrifugation at 70,000×gover 20% sucrose (wt/vol) in Tris-buffered saline (0.1 M Tris-Cl [pH7.4], 0.1 M NaCl). Concentrated virus was resuspended in Tris-bufferedsaline and stored at −80° C. Virus titration was performed by addingserial dilutions of the virus to ARPE-19 EC and by immunostaining forimmediate early-1 protein (IE1) after 48 h of incubation (60).

Antibodies:

Mouse monoclonal antibodies (MAb) 1B2, 54E11, 13B5, and 62-11 as well aspeptide-specific rabbit polyclonal antisera to the individual PCsubunits were generated previously (7, 60). Rabbit polyclonal antibodiesto vaccinia virus that were used for MVA titration were purchased fromLSBio (LS-C103289). MAb specific for HCMV IE1 (p63-27) or gH (AP86) werekindly provided by Bill Britt (University of Alabama at Birmingham) (2,43). Hybridoma supernatants of UL128 specific mouse monoclonal antibodyZ9G11 was a gift from Giuseppe Gerna (Pavia University, Italy) (18). MAb19C2 specific for the vaccinia BR5 protein was a kind gift from BernardMoss (NIAID) (42).

Plasmids:

Transfer plasm ids for inserting BAC sequences into MVA by homologousrecombination or PC subunits into MVA BAC by En passant mutagenesis (8,48, 62), were generated using standard molecular biology cloningtechniques. For inserting BAC sequences into the MVA Thymidine kinase(TK) gene by homologous recombination, a transfer vector was generatedin which pBeloBAC11 sequences—including mini-F replicon, loxP site, andcos sequences—and a GFP expression cassette with vaccinia P11 latepromoter were flanked by DNA sequences homologues to the MVA TK genelocus, as shown in FIG. 1. The pBeloBAC11 vector was obtained from NewEngland Biolabs. TK homology flanks were derived by PCR amplification ofgenomic DNA sequences of MVA 1974/NIH clone 1 that corresponded to basepairs 69313-70000 and 70001-70703 of MVA strain Acambis (Accession Nr.AY603355.1). The GFP marker with P11 promoter was derived by PCRamplification from plasmid pLW73 (63). A unique Avrll restriction sitewas introduced between the ends of the TK homology flanks to allowlinearization of the entire transfer construct, as shown in FIG. 1. Forinserting the HCMV PC subunits either all together into only oneinsertion site or into two separate insertion sites of MVA-BAC DNA by Enpassant mutagenesis (48), plasmid pGEM-T-mH5 was used as a vectorbackbone to generate three different transfer constructs that comprisedcodon-optimized, P2A-linked PC subunit subsets of either UL128/130/131A(#1), gH/gL (#2) or gH/gL with an additional P2A sequence preceding thegH gene sequence (#3) (62). Generating a transfer vector comprisingP2A-linked sequences of all five PC subunits was not successful. Each ofthe PC subunit subsets within the different transfer vectors wereflanked upstream with Kozak and vaccinia mH5 promoter sequences anddownstream with a vaccinia transcription termination signal (TTTTTAT,SEQ ID NO:6) (62). In addition, kanamycin expression cassettes withadjacent I-Scel homing endonuclease restriction sites and flanking 50 bpgene duplications were introduced into the PC subunit subsets to allowEn passant-mediated BAC insertion (48, 62). Codon-optimized, P2A-linkedDNA sequences of the PC subunit were generated by taking advantage ofthe GenPlus Economy Synthesis Service from Genescript. All PC subunitgene sequences were based on HCMV strain TB40/E (TB40/E-BAC; AccessionNr. EF999921) (44), which were codon-optimized for vaccinia virus usingthe online Codon Optimization Tool from Integrated DNA Technologies(IDT). While retaining vaccinia codon usage, codon-optimized PC genesequences were silently mutated to remove runs of more than threenucleotides of the same type in a row that are known to causeinstability within heterologous DNA sequences inserted into MVA (63).Gene internal Kanamycin/I-Scel cassettes flanked by 50 bp geneduplications were generated by PCR amplification of plasmid pEPkan-Susing primers that provided 50 bp gene duplications, and insertion ofthe resulting PCR products into unique restriction sites within the PCsubunit gene sequences (49). For inserting mRFP expression cassettesinto MVA-BAC DNA, an mRFP transfer construct for En passant mutagenesiswas generated within the pGEM-T-mH5 vector analogous to the transferconstructs for the PC subunits. The mRFP gene sequence with internalkanamycin expression cassette and a 50 bp gene duplication was derivedby PCR amplification of pEP-mRFP-in (49). Detailed sequence mapsgenerated by Vector NTI (Invitrogen) for all plasmids are available. Alltransfer constructs were confirmed by sequencing.

BAC Construction:

Construction of the novel MVA-BAC with vector sequences inserted intothe TK gene locus, termed MVA-BAC^(TK), was generated by using aprocedure similar to that described previously by Domi and colleagues(13). Briefly, 70-90% confluent CEF cells were infected with MVA1974/NIH clone 1 at 0.01 MOI and after 2 h of incubation transfectedwith 2 μg of AvrII-linearized BAC transfer vector using Fugene HDtransfection reagent (Roche) according to the manufacturers instruction.MVA recombinants were isolated following six rounds of plaquepurification on CEF cells using GFP expression of the inserted BACvector as a marker. CEF cells (70-90% confluent) were then infected withthe isolated MVA recombinants at 5 MOI. After 2 h of incubation, theinfected cells were incubated in growth medium containing 45 μMIsatin-β-thiosemicarbazone (IβT) to inhibit viral hairpin resolution andto promote heat-to-tail genome concatemerization and circularization(13). Plasmid transfection of pCICre expressing Cre recombinase thatcould have potentially enhanced genome circularization by recombinationof loxP sites within the BAC sequences was not employed due tounfavorable findings made for MVA BAC generation by others previously(8, 13). After 5 h of incubation in presence of IβT, DNA was isolatedfrom the infected CEF cells using the DNAeasy Blood and Tissue genomeisolation Kit from Qiagen according to the manufacturer's instructions,and purified DNA was transformed into DH10B E. coli cells (MAXEfficiency DH10B Competent Cells, Invitrogen).

BAC Mutagenesis:

HCMV PC subunit subsets and mRFP expression cassettes were inserted intoMVA-BAC DNA by En passant mutagenesis in GS1783 bacteria cells asdescribed previously (48, 62). Briefly, transfer constructs for the HCMVand mRFP gene sequences as described above were amplified with primerscontaining 50 bp extensions homologous to target site (primers shown inTable 1), and inserted into the MVA genome via an initial Redrecombination.

TABLE 1 Genome Target posi- Primer pairs for recombination (5′ to 3′)¹site² tion³AAAAAATATATTATTTTTATGTTATTTTGTTAAAAATAATCATCGAATACGAACTAGTATAAAAAGGCGCGCCDel2  20721GAAGATACCAAAATAGTAAAGATTTTGCTATTCAGTGGACTGGATGATTCAAAAATTGAAAATAAATACAAAGGTTCAATTGTACTTTGTAATATAATGATATATATTTTCACTTTATCTCATTTGATTTTTATAAAAATTGAAAATAAATACAAAG 64/65  56741 GTTCATTCCGAAATCTGTACATCATGCAGTGGTTAAACAAAAACATTTTTATTCCTAGTATAAAAAGGCGCGCCATATGAATATGATTTCAGATACTATATTTGTTCCTGTAGATAATAACTAAAAATTTTTATCTAGTATAAAAAGGCGCGCC 69/70  63812GGAAAATTTTTCATCTCTAAAAAAAGATGTGGTCATTAGAGTTTGATTTTTATAAAAATTGAAAATAAATACAAAGGTTCTTGGGGAAATATGAACCTGACATGATTAAGATTGCTCTTTCGGTGGCTGGTAAAAAATTGAAAATAAATACAAAGGTTCDel3 149341ACAAAATTATGTATTTTGTTCTATCAACTACCTATAAAACTTTCCAAATACTAGTATAAAAAGGCGCGCCGGTTTATTGGATTCGTGTAATCATATATTTTGCATAACATGCATCATTTTTATAAAAATTGAAAATAAATACAAAGGTTC  7/8  12802ACAATTATCCGACGCACCGGTTTCTCTTCGTGTTCTATGCCATATATTGATTTTTATCTAGTATAAAAAGGCGCGCCGAATATGACTAAACCGATGACCATTTAAAAACCCCTCTCTAGCTTTCACTAAAAATTGAAAATAAATACAAAGGTTC 44/45  37330ATAATGTTTTTATATTATACATGTTCTAAAAGAATAATCGATACAGTTTACTAGTATAAAAAGGCGCGCCGTTCGCGGCTAATCGCGATAATGTAGCTTCTAGACTTTTGTCCTAATTTTTATAAAAATTGAAAATAAATACAAAGGTTC 47/48  38924CTGGACGACACGGATTTATTAATATCGAAAAGGATATAATTGTATTTTAGTTTTTATCTAGTATAAAAAGGCGCGCCATCTAATGGATAAACTGAATCTAACAAAGAGCGACGTACAACTGTTGTAATTTTTATCTAGTATAAAAAGGCGCGCC 55/56  48516CTTTGAAAGAATGTTTGGTTCAAAACCTACATTTTACGAAGCATAATTTTTATAAAATTGAAAATAAATACAAAGGTTCGTTGTTGGCGTTGGTGGCGCTAGTCATCACATTAACTATTTTTTATTACTTTATACTATAATTTTTATAAAAATTGAAAA 81/82  72908 TAAATACAAAGGTTCTTATGGCAGGTGAGATGTTTGTTAGAAGTCAGTCTAGTACTATTATAGTATAATTTTTATCTAGTATAAAAAGGCGCGCCATAAGATATCTTCTCAAAAGATCAAGGAAATGGAAGAAACAGAAGACTTTTAATTTTTATCTAGTATAAAAAGGCGCGCC 90/91  82067GTTTAAAAGACAGATCATAGAAAAATATGTTATTGATAAGAATTAATTTTTATAAAAATTGAAAATAAATACAAAGGTTCGTTATTTTATGTCACCGCATTGGTGTTCCGATTTTAGTAATATGGAATAGTTTTTATCTAGTATAAAAAGGCGCGCC 92/93  83162GCTGTTATGGTTCCTTACAGGAACATTCGTTACCGCATTTATCTAATTTTTATAAAAATTGAAAATAAATACAAAGGTTCAGGATGTTATTACGAATCATTAAAAAAATTAACTGAGGATGATTGATTTTTATAAAAATTGAAAATAAATACAAAGGTTC107/108 100231ACAATCCCGTTATAAAAATACACGATGGTAAATTAATTTATATTTTCTAATTTTTATCTAGTATAAAAAGGCGCGCCTCCATCTAGACTATATTATCAAAATTTGGAAACTTCAAAAACGATATTAGTTTTTATCTAGTATAAAAAGGCGCGCC116/117 109461AGACTTGATTGTGACATTTAGAGAACGATATTCGTATAAATTCTAATTTTTATAAAAATTGAAAATAAATACAAAGGTTCATTGTTTATACTCAAGATATTCGTTAAACGAATTAAAATTATTTAATTTTTATAAAAATTGAAAATAAATACAAAGGTTC122/123 117576AGGAACAGATTAATCCAGACGATTGTTGTCTGGATATGGGAATGTATTAATTTTTATCTAGTATAAAAAGGCGCGCCGTTACCTCCGCAGTTTTTACGAGCGATTTCACGTTCAGCCTTCATGCGTCTTTTTATCTAGTATAAAAAGGCGCGCCDel6 129963GTGACAGAAGCTAAACCCGATAACGATAAGCGAATTCATGCTATAATTTTTATAAAAATTGAAAATAAATACAAAGGTTCATTGATAATATAAATATGAGCATTAGTATTTCTGTGGATTAATAGATTTTTATAAAAATTGAAAATAAATACAAAGGTTC148/149 137500TTATGAGGTATTTAGAGATTAGAGATGATTAATGATCCCCATACTAGAAATTTTTATCTAGTATAAAAAGGCGCGCC¹Undelined sequences indicate 5′ primer extensions that mediatedrecombination. ²Recombination target sites given either as MVA deletionsites (Del2, Del3, and Del6) or as intergenic regions between theindicated ORFs of MVA (Accession Nr. U94848). ³Recombination insertionposition within the MVA genome (Accession NR. U94848).

Subsequently, the kanamycin marker within the gene sequences wasseamlessly removed by a second Red recombination utilizing theengineered 50 bp gene duplications flanking the marker sequences (49,62). For inserting P2A-linked PC subunits into two separate MVAinsertion sites (MVA^(TK)-PC2A2), the UL128/130/131A and gH/gL subunitsubsets were successively inserted by two subsequent En passantreactions into the MVA Deletion 2 site (Del2) and the intergenic regionbetween MVA genes 69 and 70 (IGR69/70; Accession Nr. U94848),respectively. For inserting the PC subunits all together into only oneinsertion site (MVA^(TK)-PC2A1 and MVA^(Del3)-PC2A1), the UL128/130/131Asubunits were inserted into the IGR69/70 by a first En passantmutagenesis reaction, and the gH/gL subunits were subsequently insertedinto the UL128/130/131A-containing IGR69/70 site by a second En passantrecombination reaction. All sequences inserted into the MVA-BAC DNA andsequences that mediated recombination were verified by sequencing.Detailed sequence maps are available for all engineered recombinantMVA-BAC constructs.

Virus Reconstitution:

Virus reconstitution from BAC DNA was performed as previously described(8, 60). Briefly, BHK cells were seeded at 1×10⁵ cells per well in a sixwell plate and 16-20 h later transfected with ˜2-4 μg of BAC DNA thatwas prepared from E. coli by alkaline lysis (4). Transfection wasperformed using X-tremeGENE HP DNA transfection reagent (Roche)according to manufacturer's instructions. Four hours post-transfection,BHK cells were infected with FPV at 0.1 MOI to initiate virustranscription. Virus reconstitution was monitored by GFP expression ofthe BAC vector, and transfected BHK cells were re-seeded in a 1:2 ratiountil 100% of cells were infected.

Western Blot:

Immunoblot detection of the HCMV PC subunits expressed from the MVArecombinants was performed by standard procedures. Briefly, 80-90%confluent BHK cells were infected with the MVA recombinants at MOI 5 andgrown overnight for 16-20 h. Infected cells were harvested, clarifiedfrom cell debris by centrifugation at 300×g, and lysed in SDS samplebuffer (2% SDS, 100 mM dithiothreitol [DTT], and 125 mM Tris-HCl [pH8.8]). Proteins were boiled, electrophoretically separated by SDS-PAGE,and blotted to a PVDF membrane. HCMV gL and UL131A were detected withrabbit polyclonal antisera diluted 1/3000; UL128 and gH were detectedwith anti-HCMV UL128 MAb Z9G11 or gH MAb AP86 at a final concentrationof 0.1 and 10 μg/ml, respectively; and UL130 and vaccinia BR5 weredetected by using hybridoma supernatants diluted 1/10. Proteins werevisualized with secondary antibodies (anti-rabbit or anti-mouse IgGantibody, Sigma) coupled to horseradish peroxidase (HRP) andchemiluminescence detection (Pierce ECL WB substrate, Pierce).

Flow Cytometry:

Cell surface Flow cytometry staining to detect HCMV PC subunitsexpressed from the different MVA vectors by PC-specific NAb wasperformed as described previously (7). Briefly, BHK-21 (70-90%confluent) were infected with the MVA vectors at MOI 5. At 4 h postinfection, infected cells were collected, washed in phosphate bufferedsaline (PBS), and incubated for 1 h at 4° C. with 10 μg/ml NAb. Afterwashing with PBS, the cells were incubated with Alexa Fluor 647 goatanti-mouse IgG (Life Technologies) at a dilution of 1:2,000. The cellswere washed again and resuspended in PBS with 0.1% bovine serum albumin(BSA). Fifteen thousand events were collected using a Gallios flowcytometer (Beckman Coulter) and analyzed with FlowJo software (TreeStar).

Replication Kinetics:

Multi-step replication kinetics to investigate the growth properties ofMVA-BAC^(TK)-derived virus (MVA™) in comparison to wild-type MVA(MVA^(WT); MVA 1974/NIH clone 1) was performed as follows. CEF cells of70-90% confluency seeded in 6-well plates were infected induplicate-wells per virus and per harvesting time point with eitherMVA^(TK) or MVA^(WT) at 0.05 MOI and harvested every 12 h for a periodof 72 h. Collected cells were resuspended in 1 ml of Minimum EssentialMedium (Corning) containing 2% Fetal bovine albumin. Virus was releasedfrom infected cells by standard thaw/freeze and sonication techniques.Virus prepared from each well and inoculum virus used to infect the CEFcells (time point 0) were titrated on BHK cells in duplicates todetermine the titer for each time point based on four independent countsof viral foci per virus and per time point.

BAC Sequencing:

Sequencing of MVA-BAC^(TK) (#1-81) was performed by Illumina sequencingat the Integrative Genomics Core Services of the City of Hope.MVA-BAC^(TK) DNA (250 ng) was fragmented by using Covaris S220 with the300 bp peak setting. The fragmented DNA was end-repaired and ligated toIllumina adaptor oligonucleotides with TruSeq DNA LT Sample Prep kit(Illumina). Ligation products were purified with 1.0× Ampure XP beads(Beckman Coulter). The purified products were used for clustergeneration on cBot cluster generation system with HiSeq PE Cluster KitV3 (Illumina). Sequencing run was performed in the paired end mode of101 cycles of read1, 7 cycles of index read and 101 cycles of read2using HiSeq2500 platform with HiSeq SBS Kit V3 (Illumina). Real-timeanalysis (RTA) 2.2.38 software was used to process the image analysisand base calling.

Mouse Immunization:

The Institutional Animal Care and Use Committee (IACUC) of the BeckmanResearch Institute of City of Hope approved protocol 98004 assigned forthis study. All study procedures were carried out in strict accordancewith the recommendations in the Guide for the Care and Use of LaboratoryAnimals and the Public Health Service Policy on the Humane Care and Useof Laboratory Animals. Methods of euthanasia followed “Report of theAVMA Panel on Euthanasia”(avma.ora/kb/policies/documents/euthanasia.pdf). BALB/c mice (JacksonLaboratory) were vaccinated twice in three weeks interval viaintraperitoneal (i.p.) route with 5×10⁷ PFU of MVA. Blood samples fordetermining serum NAb titers were collected by eye bleed.

Neutralization Assay:

HCMV microneutralization assay was performed similar to publishedreports (7, 60). Heat-inactivated sera were serially two-fold diluted in100 μl volumes using complete growth medium for ARPE-19 EC or MRC-5 FBdepending on the cell type used in the assay. Dilutions ranged from 1:25to 1:102400. Diluted serum was mixed with 100 μl of complete growthmedium containing approximately 2400 PFU of HCMV TB40/E. After 2 hincubation, virus/sera mixtures were added in triplicate (50 μl) toARPE-19 or MRC-5 cells seeded the day before at 1.5×10⁴ cells/well in aclear bottom polystyrene 96-well plate (Corning) that contained 50 μlper well of complete growth medium. Cells were grown for 48 h and fixedin methanol/acetone. Infected cells were identified by immunostainingusing mouse anti-HCMV IE1 Ab (p63-27) and the Vectastain ABC kit(VectorLabs). The substrate was 3, 3′-diaminobenzidine (DAB,VectorLabs). Plates were analyzed by an automated system using the AxioObserver Z1 inverted microscope equipped with a linear motorized stage(Carl Zeiss). IE1 positive nuclei were counted using ImagePro Premier(Media Cybernetics). For each dilution the average number of positivenuclei in triplicate was calculated. The percent neutralization titer(NT) for each dilution was calculated as follows: NT=[1−(positive nucleinumber with immune sera)/(positive nuclei number with pre-immunesera)]×100. The titers that gave 50% neutralization (NT50) werecalculated by determining the linear slope of the graph plotting NTversus plasma dilution by using the next higher and lower NT values thatwere closest to 50% neutralization.

Statistics:

GraphPad Prism software version 5.0 (GraphPad) was used to compare NAbtiters in the different vaccine groups by statistical analysis usingWilcoxon matched-pairs test.

Example 2: Construction of a Novel MVA-BAC, Termed MVA-BAC^(TK)

BAC clones of large viral genomes are powerful tools to generaterecombinant virus by highly-efficient and versatile bacterial-basedmutagenesis techniques (46). For MVA, two different BAC clones have beendescribed (8, 35). One of these BAC, the original MVA-BAC generated byCottingham and colleagues (8), has formed the basis for a previouslyintroduced vaccine concept to stimulate high-titer HCMV NAb in mice andRM based on co-expression of all five PC subunits from a single MVAvector, termed MVA-PC (60). For generating the original MVA-BAC, BACvector sequences were introduced into the MVA deletion 3 site (Del3), acommonly used insertion site for stable maintenance of heterologous DNAsequences (34). To distinguish this BAC from the newly developed MVA-BAC(MVA-BAC^(TK)), the original BAC clone is referred to as MVA-BAC^(Del3),and virus reconstituted from this BAC is designated with MVA^(Del3).Accordingly, MVA-PC will be designated herein as MVA^(Del3)-PC to referto its origin from the original MVA-BAC^(Del3) (60). The strategydisclosed herein to develop a novel MVA-BAC, termed MVA-BAC^(TK), isbased on introduction of the BAC vector into the MVA TK gene locus toretain MVA Del3 and other commonly used insertion sites (Del2, IGR64/65,IGR69/70 (32, 34, 63) accessible for transgene insertion while providingthe option to generate a seamless self-excisable BAC vector within theTK gene sequence using techniques as described previously (10, 47, 61).Based on a procedure recently introduced for the generation of avaccinia virus BAC by Domi and colleagues (13), pBeloBAC11 vectorsequences together with a GFP expression cassette were inserted into theTK gene of the MVA genome by homologous recombination in CEF cells, andcircularized genomes of plaque purified BAC recombinant virus weretransformed into DH10B E. coli cells, as shown in FIG. 1. PCR andrestriction enzyme digestion were used to identify clones potentiallyharboring full-length MVA genomes with BAC sequences inserted at the TKgene. Some of these BAC clones were additionally tested for MVAreconstitution in BHK cells to support the integrity of the cloned MVAgenomes. One BAC clone (#1-81) was ultimately selected as MVA-BAC^(TK).

Example 3 Characterization of MVA-BAC^(TK)

To further evaluate the integrity of the cloned MVA genome ofMVA-BAC^(TK) (#1-81), MVA-BAC^(TK) DNA was investigated by extensiverestriction pattern analysis and MVA-BAC^(TK)-derived virus (MVA^(TK))by multi-step growth kinetics in BHK cells. As shown in FIG. 1B, BACrestriction pattern observed for MVA-BAC^(TK) in vitro were comparableto those predicted for MVA-BAC^(TK) in silico using Vector NTI. Inaddition, MVA^(TK) showed replication kinetics in BHK cells that weresimilar to those of wtMVA 1974/NIH clone 1 (FIG. 1E). Note that overallslightly lower virus titers were observed for wtMVA than for MVA^(TK),which was most likely a result of the slightly lower amounts of inoculumvirus of wtMVA compared to MVA^(T). Based on these findings, thecomplete MVA-BAC^(TK) #1-81 was sequenced by shotgun sequencing at4000-fold coverage, resulting in a single sequence contig (excluding theflanking MVA repeat regions), and the sequence of the cloned genome ofMVA-BAC^(TK) was found to be identical to the genome sequence of MVAstrain Acambis (Accession Nr. AY603355.1). Finally, to evaluate whetherMVA-BAC^(TK) allowed to generate virus recombinants, MVA-BAC^(TK) wastransferred to GS1783 E. coli cells that support En passant mutagenesis,a highly-efficient and versatile BAC manipulation procedure (10, 48).Using this technique, an mRFP expression cassette was introduced into 12different commonly and non-commonly used insertion sites at differentpositions of the MVA genome (FIG. 1C). As summarized in Table 1, virusexpressing mRFP—and GFP of the BAC vector (FIG. 1D)—was reconstituted inBHK cells by all engineered BAC recombinants.

In sum, these results indicate that MVA-BAC^(TK) comprises afull-length, intact genomic clone of MVA that allows to reconstitutereplication-competent and to efficiently generate virus recombinants.

Example 4: Construction of MVA Expressing P2A-Cleavable HCMV PC Subunits

MVA^(Del3)-PC was generated by introducing each of the five PC subunitswith its own mH5 promoter into a different commonly used MVA insertionsite (Del2, Del3, IGR64/65, IGR69/70; FIG. 1C) to allow equal andhigh-level expression of the PC subunits while minimizing the potentialrisk of intra- or intermolecular homologous recombination betweenpromoter elements (60, 62). Because of the separate insertion of the PCsubunits, occupancy of commonly used insertion sites, and multiple mH5promoter elements, insertion of additional HCMV antigens intoMVA^(Del3)-PC might render the vector unstable or replicationincompetent. To address this issue, the ribosomal skipping mechanismmediated by 2A peptides to express polycistronic PC subunits from onlyone or two MVA insertion sites was exploited (45). Of the different,commonly used 2A peptide sequences, the 2A peptide of porcineteschovirus-1 (P2A) has been shown recently to mediate most effectivecleavage of 2A-linked polyproteins (24). Therefore, to allow efficientand equal processing of all five PC subunits and, hence, potentiallytheir stoichiometric expression, all signals linking the PC subunitswere based on P2A peptides. To prevent instability by homologousrecombination of the P2A ribosomal skipping signals, four different DNAsequences with varying codon usage were used to encode P2A peptideslinking different PC subunits (FIG. 2B). Using En passant-mediatedmutagenesis of MVA-BAC^(TK), codon-optimized, P2A-linked DNA sequencesof the five PC subunits were inserted either all together into theIGR69/70 insertion site for generating MVA expressing self-cleavablepolyproteins composed of all five PC subunits (MVA^(TK)-PC2A1, FIG. 2A),or separately as subunit subsets into the Del2 and IGR69/70 insertionsites to generate MVA co-expressing self-processing polyproteinscomposed of UL128/130/131A and gH/gL (MVA^(TK)-PC2A2, FIG. 2A). Whileboth insertion sites have been described to allow stable transgenesinsertion, the G1L/I8R insertion site is known to promote stablepropagation of especially large sequences or antigens with potentialcellular toxicity such as transmembrane proteins (53). For comparing NAbinduction by self-processing PC subunits of virus derived fromMVA-BAC^(TK) and the original MVA-BAC^(Del3), MVA with all five PCsubunits inserted into IGR69/70 using MVA-BAC^(Del3) (MVA^(Del3)-PC2A1)was also generated, which was analogously constructed to MVA^(TK)-PC2A1(FIG. 2A).

Example 5: Expression and Cleavage of P2A-Linked HCMV PC SubunitsEncoded by MVA

To characterize the expression and cleavage of the P2A-linked HCMV PCsubunits expressed from the polycistronic MVA vectors (MVA^(TK)-PC2A1,MVA^(TK)-PC2A2, and MVA^(Del3)-PC2A1), Immunoblot analysis was used todetect the PC subunits in whole cell lysates of BHK cells infected withthe different MVA vectors. As controls, MVA^(Del3)-PC and MVA^(Del3)-gBwere included in the Immunoblot analysis. As shown in FIG. 3, markeddifferences in expression of the PC subunits were observed for thedifferent MVA vectors. While robust expression of all five PC subunitscould be confirmed for MVA^(TK)-PC2A2 and control vector MVA^(Del3)-PC,expression of only four PC subunits (gH, gL, UL128, and UL130) could beverified for the pentacistronic vectors MVA^(TK)-PC2A1 andMVA^(Del3)-PC2A1. UL131A expression of MVA^(TK)-PC2A1 andMVA^(Del3)-PC2A1 could not be unambiguously verified due to highnon-specific background using polyclonal antiserum for detection.Expression levels of the PC subunits were generally higher withMVA^(TK)-PC2A2 than with MVA^(TK)-PC2A1 and MVA^(Del3)-PC2A1. Highestexpression levels of the PC subunits were observed with control vectorMVA^(Del3)-PC when compared to any of the polycistronic vectors. Thissuggests that co-expression of P2A-linked UL128/130/131A and gH/gLsubunits subsets from two separate insertion sites is more efficientthan P2A-based polycistronic expression of all five PC subunits fromonly one MVA insertion site, and highest expression is achieved when allfive PC subunits are inserted into separate MVA insertion sites.Notably, all PC subunits of the different MVA vectors were expressed bythe same vaccinia promoter (mH5, FIG. 2).

Most of the detectable PC subunits of the three polycistronic MVAvectors had higher molecular weight than their counterparts expressedfrom MVA^(Del3)-PC due to the C-terminal P2A peptide remnants followingcleavage of the PC subunits (FIG. 3). Only gH of all three polycistronicMVA vectors, and UL131A of MVA^(TK)-PC2 had similar molecular weightscompared to their counterparts expressed from MVA^(Del3)-PC as they didnot contain C-terminal P2A peptides due to their positioning at the 3′end of the polycistronic expression constructs. Importantly, proteinbands of sizes higher than the approximate expected molecular weight ofthe PC subunits (˜85 KDa for gH; ˜35 KDa for gL; ˜15 KDa for UL128; ˜35KDa for UL130; and ˜18 KDa for UL131A) that would indicate incompleteP2A-mediated cleavage were not observed, suggesting that all HCMV PC ofthe polycistronic vectors were efficiently processed. The two proteinbands of different molecular weight that were observed for UL128 and gLfor all three polycistronic vectors appeared to represent immature andmature forms of UL128 or gL rather than incompletely cleaved subunits asthe observed protein bands did not match products with molecular weightthat would result from incomplete cleavage of the PC subunits. Proteinsbands that were of lower molecular weight than the anticipated sizes ofthe PC subunits as for example observed for UL130 and gL ofMVA^(TK)-PC2A2 and MVA^(Del3)-PC were most likely degradation products,though they may also have resulted from different expression orreplication properties of the MVA vectors (FIG. 3).

In sum, these results indicate that the P2A-linked PC subunits expressedfrom the three polycistronic MVA vectors are efficiently cleaved, thoughMVA^(TK)-PC2A2 expresses all five HCMV PC subunits with higher efficacythan MVA^(TK)-PC2A1 and MVA^(Del3)-PC2A1.

Example 6: Cell Surface Detection of MVA-Expressed P2A-Linked PCSubunits by NAb

A panel of PC-specific NAb from MVA^(Del3)-PC immunized mice that hadantigen recognition pattern and neutralization potency similar to humanNAb isolated from HCMV⁺ individuals was recently isolated (7, 29). Twoof these isolated NAb recognized quaternary conformational epitopesformed by UL128/130/131A (1B2) or UL130/131A (54E11), while two otherNAb recognized epitopes constituted by UL128 (13B5) or gH alone (62-11)(29). By taking advantage of these four existing NAb, it wasinvestigated by cell surface Flow cytometry staining of MVA infected BHKcells whether the PC subunits expressed from the different polycistronicMVA vectors assembled into complexes and formed conformational andlinear neutralizing epitopes. MVA^(Del3)-PC and MVA^(Del3) expressing gBwere included as controls in the Flow cytometry analysis. As shown inFIG. 4, BHK cells infected with either of the three polycistronic MVAvectors or control vector MVA^(Del3)-PC were efficiently stained withall four NAb. While the intensity of the BHK staining with all four NAbwas generally comparable for all MVA vectors; slightly lower stainingintensity was observed for the two pentacistronic vectors MVA^(TK)-PC2A1and MVA^(Del3)-PC2A1 and slightly higher staining intensity was observedfor MVA^(TK)-PC2A2 compared to MVA^(Del3)-PC.

In sum, these results provide evidence that the P2A-linked PC subunitsexpressed from all three polycistronic MVA vectors assemble efficientlyand are transported to the cell surface as five-member protein complexesthat present different conformational and linear neutralizing epitopes.

Example 7: NAb Induction by MVA Vectors Expressing P2A-Linked PCSubunits

In order to investigate whether the developed MVA vectors expressingP2A-linked PC subunits have ability to elicit HCMV specific NAbresponses, NAb induction by the polycistronic MVA vectors in vaccinatedBalb/c mice was evaluated. Groups of five or six Balb/c mice werevaccinated twice in three weeks interval with the MVA vectors, and HCMVspecific NAb responses in mouse sera were measured against HCMV strainTB40/E on ARPE-19 EC and MRC-5 FB by microneutralization assay over aperiod of 6 months. A two-dose immunization schedule was chosen based ona recent observation that two immunizations with MVA^(Del3)-PC aresufficient to stimulate high-titer HCMV specific NAb in mice and RM(60). MVA^(Del3)-PC was included as control in the immunization study.All polycistronic MVA vectors expressing P2A-linked PC subunits andcontrol vector MVA^(Del3)-PC stimulated comparable and potent EC and FBspecific NAb responses that were consistent with those observedpreviously with MVA^(Del3)-PC in mice (60). Robust NAb responses wereinduced by all MVA vectors after only one immunization, and theseresponses were boosted in all vaccine groups to maximum titers after thesecond immunization. In addition, NAb remained relatively stable in allvaccine groups until the end of the experiment at week 24 after thebooster immunization. Consistent with previous immunization studiesbased on the PC and with NAb responses found in HCMV⁺ individuals, NAbinduced by all MVA vectors that were measured on MRC-5 FB weresignificantly lower than those measured on ARPE-19 EC (11, 18, 23, 60).While EC specific NAb titers across all vaccine groups were generallycomparable (except at week 24 comparing MVA^(Del3)-PC andMVA^(Del3)-PC2A1), FB specific NAb titers were generally slightly lowerin the MVA^(Del3)-PC control vector group than in animal groupsimmunized with any of the polycistronic MVA vectors (except at week 24comparing MVA^(Del3)-PC and MVA^(TK). PC2A1). Notably, NAb responsesthat were measured in this study for control vector MVA^(Del3)-PCappeared slightly lower than those observed in mice with MVA^(Del3)-PCin previous studies (60).

In sum, these results indicate that all three MVA vectors expressingP2A-linked PC subunits (MVA^(TK)-PC2A2, MVA^(TK)-PC2A1, andMVA^(Del3)-PC2A1) have potent and comparable ability to elicit HCMV NAbresponses in mice.

REFERENCES

The references, patents and published patent applications listed below,and all references cited in the specification above are herebyincorporated by reference in their entirety, as if fully set forthherein.

-   1. Adler, B., L. Scrivano, Z. Ruzclcs, B. Rupp, C. Sinzger, and U.    Koszlnowskl. 2006. Role of human cytomegalovirus UL131A in cell    type-specific virus entry and release. The Journal of general    virology 87:2451-2460.-   2. Andreoni, M., M. Faircloth, L Vugler, and W. J. Britt. 1989. A    rapid microneutralization assay for the measurement of neutralizing    antibody reactive with human cytomegalovirus. Journal of virological    methods 23:157-167.-   3. Bernstein, D. I., F. M. Munoz, S. T. Callahan, R. Rupp, S. H.    Wootton, K. M. Edwards, C. B. Turley, L. R. Stanberry, S. M.    Patel, M. M. McNeal, S. Plchon, C. Amegashle, and A. R.    Bellamy. 2016. Safety and efficacy of a cytomegalovirus glycoprotein    B (gB) vaccine in adolescent girls: A randomized clinical trial.    Vaccine 34:313-319.-   4. Blmboim, H. C., and J. Doly. 1979. A rapid alkaline extraction    procedure for screening recombinant plasmid DNA. Nucleic acids    research 7:1513-1523.-   5. Butler, D. 2016. Zika raises wider birth-defect issue. Nature    835:17-17.-   6. Carroll, M. W., and B. Moss. 1997. Host range and    cytopathogenicity of the highly attenuated MVA strain of vaccinia    virus: propagation and generation of recombinant viruses in a    nonhuman mammalian cell line. Virology 238:198-211.-   7. Chluppesi, F., F. Wussow, E. Johnson, C. Blan, M. Zhuo, A.    Rajakumar, P. A. Barry, W. J. Britt, R. Chakraborty, and D. J.    Diamond. 2015. Vaccine-Derived Neutralizing Antibodies to the Human    Cytomegalovirus gH/gL Pentamer Potently Block Primary    Cytotrophoblast Infection. Journal of virology 89:11884-11898.-   8. Cottingham, M. G., R. F. Andersen, A. J. Spencer, S. Saurya, J.    Furze, A. V. Hill, and S. C. Gilbert. 2008. Recombination-mediated    genetic engineering of a bacterial artificial chromosome clone of    modified vaccinia virus Ankara (MVA). PloS one 3:e1638.-   9. Cottingham, M. G., and M. W. Carroll. 2013. Recombinant MVA    vaccines: dispelling the myths. Vaccine 31:4247-4251.-   10. Cottingham, M. G., and S. C. Gilbert. 2010. Rapid generation of    markerless recombinant MVA vaccines by en passant recombineering of    a self-excising bacterial artificial chromosome. Journal of    virological methods 168:233-236.-   11. Cui, X., B. P. Meza, S. P. Adler, and M. A. McVoy. 2008.    Cytomegalovirus vaccines fail to induce epithelial entry    neutralizing antibodies comparable to natural infection. Vaccine    26:5760-5766.-   12. de Felipe, P. 2004. Skipping the co-expression problem: the new    2A “CHYSEL” technology. Genetic vaccines and therapy 2:13.-   13. Domi, A., and B. Moss. 2002. Cloning the vaccinia virus genome    as a bacterial artificial chromosome in Escherichia coli and    recovery of infectious virus in mammalian cells. Proceedings of the    National Academy of Sciences of the United States of America    99:12415-12420.-   14. Donnelly, M. L., G. Luke, A. Mehrotra, X. Li, L E. Hughes, D.    Gani, and M. D. Ryan. 2001. Analysis of the aphthovirus 2A/2B    polyprotein ‘cleavage’ mechanism indicates not a proteolytic    reaction, but a novel translational effect: a putative ribosomal    ‘skip’. The Journal of general virology 82:1013-1025.-   15. Draper, S. J., M. G. Cottingham, and S. C. Gilbert. 2013.    Utilizing poxviral vectored vaccines for antibody induction-progress    and prospects. Vaccine 31:4223-4230.-   16. Freed, D. C., Q. Tang, A. Tang, F. Li, X. He, Z. Huang, W. Meng,    L Xia, A. C. Finnefrock, E. Durr, A. S. Espeseth, D. R. Casimiro, N.    Zhang, J. W. Shiver, D. Wang, Z. An, and T. M. Fu. 2013. Pentameric    complex of viral glycoprotein H is the primary target for potent    neutralization by a human cytomegalovirus vaccine. Proceedings of    the National Academy of Sciences of the United States of America    110:E4997-5005.-   17. Gerna, G., E. Percivalle, L Perez, A. Lanzavecchia, and D.    Lilleri. 2016. Monoclonal Antibodies to Different Components of the    Human Cytomegalovirus (HCMV) Pentamer gH/gL/pUL128L and Trimer    gH/gL/gO as well as Antibodies Elicited during Primary HCMV    Infection Prevent Epithelial Cell Syncytium Formation. Journal of    virology 90:6216-6223.-   18. Gerna, G., A. Sarasini, M. Patrone, E. Percivalle, L Florina, G.    Campanini, A. Gallina, F. Baldanti, and M. G. Revello. 2008. Human    cytomegalovirus serum neutralizing antibodies block virus infection    of endothelial/epithelial cells, but not fibroblasts, early during    primary infection. The Journal of general virology 89:853-865.-   19. Gilbert, S. C. 2013. Clinical development of Modified Vaccinia    virus Ankara vaccines. Vaccine 31:4241-4246.-   20. Hahn, G., M. G. Revello, M. Patrone, E. Percivalle, G.    Campanini, A. Sarasini, M. Wagner, A. Gallina, G. Milanesi, U.    Koszinowski, F. Baldanti, and G. Gerna. 2004. Human cytomegalovirus    UL131-128 genes are indispensable for virus growth in endothelial    cells and virus transfer to leukocytes. Journal of virology    78:10023-10033.-   21. Hofmann, I., Y. Wen, C. Ciferri, A. Schulze, V. Fuhner, M.    Leong, A. Gerber, R. Gerrein, A. Nandi, A. E. Lilja, A. Carfi,    and H. Laux. 2015. Expression of the Human Cytomegalovirus Pentamer    Complex for vaccine use in a CHO system. Biotechnology and    bioengineering.-   22. Jiang, X. J., B. Adler, K. L Sampalo, M. Digel, G. Jahn, N.    Ettischer, Y. D. Stierhof, L. Scrivano, U. Koszinowski, M. Mach,    and C. Sinzger. 2008. UL74 of human cytomegalovirus contributes to    virus release by promoting secondary envelopment of virions. Journal    of virology 82:2802-2812.-   23. Kabanova, A., L Perez, D. Liller, J. Marcandalli, G. Agatic, S.    Becattini, S. Prelte, D. Fuschillo, E. Percivalle, F. Sallusto, G.    Gerna, D. Corti, and A. Lanzavecchia. 2014. Antibody-driven design    of a human cytomegalovirus gHgLpUL128L subunit vaccine that    selectively elicits potent neutralizing antibodies. Proceedings of    the National Academy of Sciences of the United States of America    111:17965-17970.-   24. Kim, J. H., S. R. Lee, L. H. Li, H. J. Park, J. H. Park, K. Y.    Lee, M. K. Kim, B. A. Shin, and S. Y. Choi. 2011. High cleavage    efficiency of a 2A peptide derived from porcine teschovirus-1 in    human cell lines, zebrafish and mice. PloS one 6:e18556.-   25. La Rosa, C., J. Longmate, J. Martinez, Q. Zhou, T. I.    Kaltcheva, W. Tsai, J. Drake, M. Carroll, F. Wussow, F.    Chiuppesi, N. Hardwick, S. Dadwal, I. Aldoss, R. Nakamura, J. A.    Zaia, and D. J. Diamond. 2016. MVA vaccine encoding CMV antigens    safely induces durable expansion of CMV-specific T-cells in healthy    adults. Blood.-   26. Lilleri, D., A. Kabanova, A. Lanzavecchia, and G. Gerna. 2012.    Antibodies against neutralization epitopes of human cytomegalovirus    gH/gL/pUL128-130-131 complex and virus spreading may correlate with    virus control in vivo. Journal of clinical immunology 32:1324-1331.-   27. Lilleri, D., A. Kabanova, M. G. Revello, E. Percivalle, A.    Sarasini, E. Genini, F. Sallusto, A. Lanzavecchia, D. Corti, and G.    Gerna. 2013. Fetal human cytomegalovirus transmission correlates    with delayed maternal antibodies to gH/gL/pUL128-130-131 complex    during primary infection. PloS one 8:e59863.-   28. Loughney, J. W., R. R. Rustandi, D. Wang, M. C. Troutman, L. W.    Dick, Jr., G. Li, Z. Liu, F. Li, D. C. Freed, C. E. Price, V. M.    Hoang, T. D. Culp, P. A. DePhillips, T. M. Fu, and S. Ha. 2015.    Soluble Human Cytomegalovirus gH/gL/pUL128-131 Pentameric Complex,    but not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents    Dominant Native Neutralizing Epitopes. The Journal of biological    chemistry.-   29. Macagno, A., N. L Bernasconi, F. Vanzetta, E. Dander, A.    Sarasini, M. G. Revello, G. Gerna, F. Sallusto, and A.    Lanzavecchia. 2010. Isolation of human monoclonal antibodies that    potently neutralize human cytomegalovirus infection by targeting    different epitopes on the gH/gL/UL128-131A complex. Journal of    virology 84:1005-1013.-   30. Manghera, A., and G. R. McLean. 2016. Human cytomegalovirus    vaccination: progress and perspectives of recombinant gB. Future    virology 11:439-449.-   31. Manicklal, S., V. C. Emery, T. Lazzarotto, S. B. Boppana,    and R. K. Gupta. 2013. The “silent” global burden of congenital    cytomegalovirus. Clinical microbiology reviews 26:86-102.-   32. Manuel, E. R., Z. Wang, Z. Li, C. La Rosa, W. Zhou, and D. J.    Diamond. 2010. Intergenic region 3 of modified vaccinia ankara is a    functional site for insert gene expression and allows for potent    antigen-specific immune responses. Virology 403:155-162.-   33. Mayr, A., and K. Malicki. 1966. [Attenuation of virulent fowl    pox virus in tissue culture and characteristics of the attenuated    virus]. Zentralblatt fur Veterinarmedizin. Reihe B. Journal of    veterinary medicine. Series B 13:1-13.-   34. Meinger-Henschel, C., M. Schmidt, S. Lukassen, B. Unke, L    Krause, S. Konletzny, A. Goesmann, P. Howley, P. Chaplin, M. Suter,    and J. Hausmann. 2007. Genomic sequence of chorioallantois vaccinia    virus Ankara, the ancestor of modified vaccinia virus Ankara. The    Journal of general virology 88:3249-3259.-   35. Meisinger-Henschel, C., M. Spath, S. Lukassen, M.    Wolferstatter, H. Kachelrless, K. Baur, U. Dirmeier, M. Wagner, P.    Chaplin, M. Suter, and J. Hausmann. 2010. Introduction of the six    major genomic deletions of modified vaccinia virus Ankara (MVA) into    the parental vaccinia virus is not sufficient to reproduce an    MVA-like phenotype in cell culture and in mice. Journal of virology    84:9907-9919.-   36. Mlakar, J., M. Korva, N. Tul, M. Popovic, M.    Poljsak-Prijatelj, J. Mraz, M. Kolenc, K. Resman Rus, T. Vesnaver    Vipotnik, V. Fabjan Vodusek, A. Vizjak, J. Pizem, M. Petrovec,    and T. Avsic Zupanc. 2016. Zika Virus Associated with Microcephaly.    The New England journal of medicine 374:951-958.-   37. Pass, R. F. 2009. Development and evidence for efficacy of CMV    glycoprotein B vaccine with MF59 adjuvant. Journal of clinical    virology: the official publication of the Pan American Society for    Clinical Virology 46 Suppl 4:S73-76.-   38. Pereira, L., M. Petitt, A. Fong, M. Tsuge, T. Tabata, J.    Fang-Hoover, E. Maidji, M. Zydek, Y. Zhou, N. Inoue, S. Loghavi, S.    Pepkowitz, L M. Kauvar, and D. Ogunyemi. 2014. Intrauterine growth    restriction caused by underlying congenital cytomegalovirus    infection. The Journal of infectious diseases 209:1573-1584.-   39. Ryan, M. D., A. M. King, and G. P. Thomas. 1991. Cleavage of    foot-and-mouth disease virus polyprotein is mediated by residues    located within a 19 amino acid sequence. The Journal of general    virology 72 (Pt 11):2727-2732.-   40. Ryckman, B. J., M. A. Jarvis, D. D. Drummond, J. A. Nelson,    and D. C. Johnson. 2006. Human cytomegalovirus entry into epithelial    and endothelial cells depends on genes UL128 to UL150 and occurs by    endocytosis and low-pH fusion. Journal of virology 80:710-722.-   41. Sampalo, K. L., Y. Cavignac, Y. D. Stierhof, and C.    Sinzger. 2005. Human cytomegalovirus labeled with green fluorescent    protein for live analysis of intracellular particle movements.    Journal of virology 79:2754-2767.-   42. Schmelz, M., B. Sodelk, M. Ericsson, E. J. Wolffe, H. Shida, G.    Hiller, and G. Grlffiths. 1994. Assembly of vaccinia virus: the    second wrapping cistema is derived from the trans Golgi network.    Journal of virology 68:130-147.-   43. Simpson, J. A., J. C. Chow, J. Baker, N. Avdalovic, S. Yuan, D.    Au, M. S. Co, M. Vasquez, W. J. Britt, and K. L Coelingh. 1993.    Neutralizing monoclonal antibodies that distinguish three antigenic    sites on human cytomegalovirus glycoprotein H have conformationally    distinct binding sites. Journal of virology 67:489-496.-   44. Sinzger, C., G. Hahn, M. Digel, R. Katona, K. L Sampalo, M.    Messerle, H. Hengel, U. Koszinowski, W. Brune, and B. Adler. 2008.    Cloning and sequencing of a highly productive, endotheliotropic    virus strain derived from human cytomegalovirus TB40/E. The Journal    of general virology 89:359-368.-   45. Szymczak, A. L., C. J. Workman, Y. Wang, K. M. Vignali, S.    Dilloglou, E. F. Vanin, and D. A. Vignali. 2004. Correction of    multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A    peptide-based retroviral vector. Nature biotechnology 22:589-594.-   46. Tischer, B. K., and B. B. Kaufer. 2012. Viral bacterial    artificial chromosomes: generation, mutagenesis, and removal of    mini-F sequences. Journal of biomedicine & biotechnology    2012:472537.-   47. Tischer, B. K., B. B. Kaufer, M. Sommer, F. Wussow, A. M. Arvin,    and N. Osterrieder. 2007. A self-excisable infectious bacterial    artificial chromosome clone of varicella-zoster virus allows    analysis of the essential tegument protein encoded by ORF9. Journal    of virology 81:13200-13208.-   48. Tischer, B. K., G. A. Smith, and N. Osterrieder. 2010. En    passant mutagenesis: a two step markerless red recombination system.    Methods Mol Biol 634:421-430.-   49. Tischer, B. K., J. von Einem, B. Kaufer, and N.    Osterrieder. 2006. Two-step red-mediated recombination for versatile    high-efficiency markerless DNA manipulation in Escherichia coli.    BioTechniques 40:191-197.-   50. Vanarsdall, A. L., P. W. Howard, T. W. Wisner, and D. C.    Johnson. 2016. Human Cytomegalovirus gH/gL Forms a Stable Complex    with the Fusion Protein gB in Virions. PLoS pathogens 12:e1005564.-   51. Vanarsdall, A. L., and D. C. Johnson. 2012. Human    cytomegalovirus entry into cells. Current opinion in virology    2:37-42.-   52. Verheust, C., M. Goossens, K. Pauwels, and D. Breyer. 2012.    Biosafety aspects of modified vaccinia virus Ankara (MVA)-based    vectors used for gene therapy or vaccination. Vaccine 30:2623-2632.-   53. Wagner, S., M. L. Bader, D. Drew, and J. W. de Ger. 2006.    Rationalizing membrane protein overexpression. Trends in    biotechnology 24:364-371.-   54. Wang, D., D. C. Freed, X. He, F. Li, A. Tang, K. S. Cox, S. A.    Dubey, S. Cole, M. B. Medi, Y. Liu, J. Xu, Z. Q. Zhang, A. C.    Finnefrock, L. Song, A. S. Espeseth, J. W. Shiver, D. R. Casimiro,    and T. M. Fu. 2016. A replication-defective human cytomegalovirus    vaccine for prevention of congenital infection. Science    translational medicine 8:362ra145.-   55. Wang, D., and T. Shenk. 2005. Human cytomegalovirus virion    protein complex required for epithelial and endothelial cell    tropism. Proceedings of the National Academy of Sciences of the    United States of America 102:18153-18158.-   56. Wang, Z., C. La Rosa, R. Maas, H. Ly, J. Brewer, S.    Mekhoubad, P. Daftarian, J. Longmate, W. J. Britt, and D. J.    Diamond. 2004. Recombinant modified vaccinia virus Ankara expressing    a soluble form of glycoprotein B causes durable immunity and    neutralizing antibodies against multiple strains of human    cytomegalovirus. Journal of virology 78:3965-3976.-   57. Wang, Z., J. Martinez, W. Zhou, C. La Rosa, T. Srivastava, A.    Dasgupta, R. Rawal, Z. Li, W. J. Britt, and D. Diamond. 2010.    Modified H5 promoter improves stability of insert genes while    maintaining immunogenicity during extended passage of genetically    engineered MVA vaccines. Vaccine 28:1547-1557.-   58. Wen, Y., J. Monroe, C. Linton, J. Archer, C. W. Beard, S. W.    Barnett, G. Palladino, P. W. Mason, A. Carfi, and A. E. Lilja. 2014.    Human cytomegalovirus gH/gL/UL128/UL130/UL131A complex elicits    potently neutralizing antibodies in mice. Vaccine 32:3796-3804.-   59. Wille, P. T., A. J. Knoche, J. A. Nelson, M. A. Jarvis,    and D. C. Johnson. 2010. A human cytomegalovirus gO-null mutant    fails to incorporate gH/gL into the virion envelope and is unable to    enter fibroblasts and epithelial and endothelial cells. Journal of    virology 84:2585-2596.-   60. Wussow, F., F. Chiuppesi, J. Martinez, J. Campo, E. Johnson, C.    Flechsig, M. Newell, E. Tran, J. Ortiz, C. La Rosa, A. Herrmann, J.    Longmate, R. Chakraborty, P. A. Barry, and D. J. Diamond. 2014.    Human cytomegalovirus vaccine based on the envelope gH/gL pentamer    complex. PLoS pathogens 10:e1004524.-   61. Wussow, F., H. Fickenscher, and B. K. Tischer. 2009.    Red-mediated transposition and final release of the mini-F vector of    a cloned infectious herpesvirus genome. PloS one 4:e8178.-   62. Wussow, F., Y. Yue, J. Martinez, J. D. Deem, J. Longmate, A.    Herrmann, P. A. Barry, and D. J. Diamond. 2013. A vaccine based on    the rhesus cytomegalovirus UL128 complex induces broadly    neutralizing antibodies in rhesus macaques. Journal of virology    87:1322-1332.-   63. Wyatt, L S., P. L Earl, W. Xiao, J. L Americo, C. A. Cotter, J.    Vogt, and B. Moss. 2009. Elucidating and minimizing the loss by    recombinant vaccinia virus of human immunodeficiency virus gene    expression resulting from spontaneous mutations and positive    selection. Journal of virology 83:7176-7184.-   64. Zhou, M., J. M. Lanchy, and B. J. Ryckman. 2015. Human    cytomegalovirus gH/gL/gO promotes the fusion step of entry into all    cell types whereas gH/gL/UL128-131 broadens virus tropism through a    distinct mechanism. Journal of virology.

What is claimed is:
 1. An expression system comprising one or morevectors, each vector comprising one or more expression cassette(s), eachexpression cassette comprising: a single promoter; two or more nucleicacid sequences that encode two or more herpesvirus glycoprotein complexsubunits or antigenic fragments thereof, wherein the two or moreherpesvirus glycoprotein complex subunits or antigenic fragmentsthereof, when expressed by the expression system, form a herpesvirusglycoprotein complex that is functional and effectively elicitsneutralizing antibody responses, and wherein the two or more herpesvirusglycoprotein complex subunits or antigenic fragments thereof are derivedfrom varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Kaposi'sSarcoma-associated herpesvirus (KSHV), human herpesvirus 6 (HHV-6),human herpesvirus 7 (HHV-7); one or more 2A peptide signal sequencesthat mediate ribosomal skipping or one or more internal ribosomal entrysites between each of the two or more nucleic acid sequences; whereinthe two or more herpesvirus glycoprotein complex subunits or antigenicfragments thereof are simultaneously expressed by the expression system;wherein the one or more vectors is (i) a plasmid or (ii) a viral vectorselected from a CMV vector, a vaccinia vector, or an adenoviral vector.2. The expression system of claim 1, wherein the one or more 2A signalsequence encode a 2A peptide of foot-and-mouth disease virus (F2A), a 2Apeptide of equine rhinitis A virus (E2A), a 2A peptide of porcineteschovirus-1 (P2A), a 2A peptide of cytoplasmic polyhedrosis virus(BmCPV 2A), a 2A peptide of flacherie virus (BmIFV 2A), or a 2A peptideof Thosea asigna virus (T2A).
 3. The expression system of claim 1,wherein the expression cassette further comprises a furin cleavage siteupstream of each of the one or more 2A signal sequences.
 4. Theexpression system of claim 1, wherein the promoter is a single promotersequence located upstream of the subunits or the antigenic fragmentsthereof.
 5. The expression system of claim 1, wherein the promoter is avaccinia virus mH5 promoter, a pSyn promoter, a p7.5 promoter, or a p11promoter.
 6. The expression system of claim 1, wherein the two or moreherpesvirus glycoprotein complex subunits include gD of HSV, gp350/220of EBV, gpK8.1 of KSHV, and accessory glycoproteins of otherherpesviruses.
 7. The expression system of claim 1, wherein the nucleicacid sequence encodes EBV EBNA1 or LMP2, or KSHV LANA, or otherimmunodominant antigens or latency associated proteins.
 8. Theexpression system of claim 1, wherein the expression system comprises: afirst vector comprising a first expression cassette, the firstexpression cassette comprising a first promoter, two or more nucleicacid sequences that encode two or more amino acids herpesvirusglycoprotein complex subunits or antigenic fragments thereof, and one ormore 2A signal sequences between each of the two or more nucleic acidsequences, wherein the two or more herpesvirus glycoprotein complexsubunits or antigenic fragments thereof, when expressed by theexpression system, form a herpesvirus glycoprotein complex; wherein thetwo or more herpesvirus glycoprotein complex subunits or antigenicfragments thereof are derived from varicella-zoster virus (VZV),Epstein-Barr virus (EBV), Kaposi's Sarcoma-associated herpesvirus(KSHV), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7); andwherein the first vector is (i) a plasmid or (ii) a viral vectorselected from a CMV vector, a vaccinia vector, or an adenoviral vector;and a second vector comprising a second expression cassette, the secondexpression cassette comprising a second promoter, two or more nucleicacid sequences that encode two or more amino acids herpesvirusglycoprotein complex subunits or antigenic fragments thereof, and one ormore 2A signal sequences between each of the two or more nucleic acidsequences, wherein the two or more herpesvirus glycoprotein complexsubunits or antigenic fragments thereof, when expressed by theexpression system, form a herpesvirus glycoprotein complex; wherein thetwo or more herpesvirus glycoprotein complex subunits or antigenicfragments thereof are derived from varicella-zoster virus (VZV),Epstein-Barr virus (EBV), Kaposi's Sarcoma-associated herpesvirus(KSHV), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7); andwherein the second vector is (i) a plasmid or (ii) a viral vectorselected from a CMV vector, a vaccinia vector, or an adenoviral vector;wherein the two or more herpesvirus glycoprotein complex subunits orantigenic fragments thereof of the first and second vectors aresimultaneously expressed by the expression system.
 9. The expressionsystem of claim 1, wherein each vector further comprises a secondexpression cassette comprising: a second promoter; two or more nucleicacid sequences that encode two or more herpesvirus glycoprotein complexsubunits or antigenic fragments thereof, wherein the two or moreherpesvirus glycoprotein complex subunits or antigenic fragmentsthereof, when expressed by the expression system, form a herpesvirusglycoprotein complex; one or more 2A signal sequences between each ofthe two or more nucleic acid sequences; wherein the two or moreherpesvirus glycoprotein complex subunits or antigenic fragments thereofof the first expression cassette and the two or more herpesvirusglycoprotein complex subunits or antigenic fragments thereof of thesecond expression cassette are simultaneously expressed by theexpression system; and wherein the two or more herpesvirus glycoproteincomplex subunits or antigenic fragments thereof of each expressioncassette are derived from varicella-zoster virus (VZV), Epstein-Barrvirus (EBV), Kaposi's Sarcoma-associated herpesvirus (KSHV), humanherpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7.
 10. The expressionsystem of claim 1, wherein the two or more nucleic acid sequences arenaturally occurring DNA sequences.
 11. The expression system of claim 1,wherein the two or more nucleic acid sequences are codon-optimized. 12.An expression system comprising a recombinant bacterial artificialchromosome (BAC) construct comprising a BAC vector and a viral vectorgenome, the viral vector genome comprising at least one expressioncassette, wherein each expression cassette comprises: a single promoter;two or more nucleic acid sequences that encode two or more herpesvirusglycoprotein complex subunits or antigenic fragments thereof, whereinthe two or more herpesvirus glycoprotein complex subunits or antigenicfragments thereof, when expressed by the expression system, form aherpesvirus glycoprotein complex; and wherein the two or moreherpesvirus glycoprotein complex subunits or antigenic fragments thereofare derived from varicella-zoster virus (VZV), Epstein-Barr virus (EBV),Kaposi's Sarcoma-associated herpesvirus (KSHV), human herpesvirus 6(HHV-6), human herpesvirus 7 (HHV-7); one or more 2A signal sequencesbetween each of the two or more nucleic acid sequences; wherein the twoor more herpesvirus glycoprotein complex subunits or antigenic fragmentsthereof are simultaneously expressed by the expression system; andwherein the viral vector is selected from a CMV vector, a vacciniavector, or an adenoviral vector.
 13. The expression system of claim 12,wherein the expression system comprises: a first vector comprising afirst expression cassette, the first expression cassette comprising afirst promoter, two or more nucleic acid sequences that encode two ormore amino acids herpesvirus glycoprotein complex subunits or antigenicfragments thereof, and one or more 2A signal sequences between each ofthe two or more nucleic acid sequences, wherein the two or moreherpesvirus glycoprotein complex subunits or antigenic fragmentsthereof, when expressed by the expression system, form a herpesvirusglycoprotein complex; wherein the two or more herpesvirus glycoproteincomplex subunits or antigenic fragments thereof are derived fromvaricella-zoster virus (VZV), Epstein-Barr virus (EBV), Kaposi'sSarcoma-associated herpesvirus (KSHV), human herpesvirus 6 (HHV-6),human herpesvirus 7 (HHV-7); and wherein the first vector is (i) aplasmid or (ii) a viral vector selected from a CMV vector, a vacciniavector, or an adenoviral vector; and a second vector comprising a secondexpression cassette, the second expression cassette comprising a secondpromoter, two or more nucleic acid sequences that encode two or moreamino acids herpesvirus glycoprotein complex subunits or antigenicfragments thereof, and one or more 2A signal sequences between each ofthe two or more nucleic acid sequences, wherein the two or moreherpesvirus glycoprotein complex subunits or antigenic fragmentsthereof, when expressed by the expression system, form a herpesvirusglycoprotein complex; wherein the two or more herpesvirus glycoproteincomplex subunits or antigenic fragments thereof are derived fromvaricella-zoster virus (VZV), Epstein-Barr virus (EBV), Kaposi'sSarcoma-associated herpesvirus (KSHV), human herpesvirus 6 (HHV-6),human herpesvirus 7 (HHV-7); and wherein the second vector is (i) aplasmid or (ii) a viral vector selected from a CMV vector, a vacciniavector, or an adenoviral vector; wherein the two or more herpesvirusglycoprotein complex subunits or antigenic fragments thereof of thefirst and second vectors are simultaneously expressed by the expressionsystem.
 14. The expression system of claim 1, wherein the two or morenucleic acid sequences are DNA sequences.
 15. The expression system ofclaim 12, wherein the two or more nucleic acid sequences are DNAsequences.
 16. The expression system of claim 1, wherein the vector is amodified vaccinia Ankara (MVA) vector.
 17. The expression system ofclaim 12, wherein the vector is a modified vaccinia Ankara (MVA) vector.